Jing Sun1,2, Yarong Mu2, Yuanyuan Jiang2, Ruilong Song2, Jianxin Yi3, Jingsong Zhou3, Jun Sun4, Xinan Jiao2,5, Richard A Prinz6, Yi Li7, Xiulong Xu8,9,10,11. 1. Institute of Comparative Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China. 2. College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China. 3. Department of Physiology, Kansas City University of Medicine and Biosciences, Kansas City, MO, 64106, USA. 4. Department of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA. 5. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China. 6. Department of Surgery, NorthShore University Health System, Evanston, IL, 60201, USA. 7. Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX, 77030, USA. 8. Institute of Comparative Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China. xxl@yzu.edu.cn. 9. College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China. xxl@yzu.edu.cn. 10. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, Jiangsu Province, China. xxl@yzu.edu.cn. 11. Department of Cell and Molecular Medicine Rush University Medical Center, Chicago, IL, 60612, USA. xxl@yzu.edu.cn.
Abstract
Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70 S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPKT172 and ULK1S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1G93A protein aggregates with autophagosomes and accelerated SOD1G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.
Autophagy plays a central role in degrading misfolded proteins such as mutated superoxide dismutase 1 (SOD1), which forms aggregates in motor neurons and is involved in the pathogenesis of amyotrophic lateral sclerosis (ALS). Autophagy is activated when UNC-51-like kinase 1 (ULK1) is phosphorylated at S555 and activated by AMP-activated protein kinase (AMPK). Autophagy is suppressed when ULK1 is phosphorylated at S757 by the mechanistic target of rapamycin (mTOR). Whether p70S6 kinase 1 (S6K1), a serine/threonine kinase downstream of mTOR, can also regulate autophagy remains uncertain. Here we report that inhibition of S6K1 by A77 1726, the active metabolite of an anti-inflammatory drug leflunomide, induced mTOR feedback activation and ULK1S757 phosphorylation in NSC34 cells, a hybrid mouse motoneuron cell line. Unexpectedly, A77 1726 did not suppress but rather induced autophagy by increasing AMPKT172 and ULK1S555 phosphorylation. Similar observations were made with PF-4708671, a specific S6K1 inhibitor, or with S6K1 siRNA. Further studies showed that A77 1726 induced AMPK phosphorylation by activating the TGF-β-activated kinase 1 (TAK1). Functional studies revealed that A77 1726 induced co-localization of mutant SOD1G93A protein aggregates with autophagosomes and accelerated SOD1G93A protein degradation, which was blocked by inhibition of autophagy through autophagy-related protein 7 (ATG7) siRNA. Our study suggests that S6K1 inhibition induces autophagy through TAK1-mediated AMPK activation in NSC34 cells, and that blocking S6K1 activity by a small molecule inhibitor such as leflunomide may offer a new strategy for ALS treatment.
Amyotrophic lateral sclerosis (ALS) is the most common form of adult-onset motoneuron degenerative disease characterized by the selective loss of motoneurons in the ventral horn of the spinal cord, the cerebral cortex, and brainstem nuclei[1, 2]. Approximately 90% of ALS is sporadic and does not have an apparent genetic linkage. The remaining 10% is familial and these patients carry a mutant gene[3]. Superoxide dismutase 1 (SOD1) was the first mutated gene to be discovered in familial ALS about two decades ago[4-6]. Mutant SOD1 proteins are prone to misfolding and forming aggregates in motoneurons. Several other genes, including TAR DNA-binding protein 43 (TDP-43), Fused in Sarcoma/Translocated in Sarcoma (FUS/TLS), and chromosome 9 open reading frame 72 (C9ORF72), have also been found to be mutated in familial ALSpatients[3]. The products of these genes, TDP-43, FUS, and DPR (dipeptide repeat), can also form aggregates that cannot be easily degraded. The presence of protein aggregates in the cytosol activates macroautophagy (often referred as autophagy), a cellular process involved in degrading long-lived proteins and damaged organelles such as mitochondria[3, 7, 8]. Inability to remove protein aggregates leads to cell death and neurodegeneration[3, 7, 8]. Recent studies have shown that several genes involved in autophagy, including p62/SQSTM1 (SQSTM1), ubiquilin 2 (UBQLN2), optineurin 1 (OPTN1), TANK-binding kinase 1 (TBK1), are mutated in familial ALSpatients[8-10]. Therapeutic intervention to activate autophagy and subsequently decrease the load of protein aggregates and oligomers has alleviated ALS in preclinical studies[8-10]. Better understanding of the regulation of autophagy will help designing novel therapeutic strategies to treat this fatal disease.Autophagy is initiated by the class III PI-3 kinase (Vps34) that complexes with Beclin-1 and ATG14 to trigger the nucleation of the membrane from the endoplasmic reticulum[11]. On the other hand, autophagy is inhibited by activation of the class I PI-3 kinase pathway through mTOR, a serine/threonine kinase that phosphorylates ULK1/2 and inhibits the assembly of the autophagic machinery (Fig. 1m)[11]. AMPK activation due to energy stress leads to ULK1S555 phosphorylation and activation, thus directly initiating autophagy[12-14]. mTOR inhibitors and AMPK activators have been sought as autophagy inducers to degrade protein aggregates in motor neurons and to ameliorate ALS progression[8]. S6K1 is a serine/threonine kinase phosphorylated and activated by mTOR, and is overexpressed and highly activated in the spinal cord of ALSpatients and in transgenic mice with the SOD1G93A gene[15, 16]. Whether S6K1 inhibition can induce autophagy and accelerate mutant SOD1 protein degradation has not been studied.
Fig. 1
The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and autophagy.
a–f The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 h (a, c, d) or were incubated in the presence of A77 1726 (200 μM) for the indicated time (b, e, f). Rapamycin (50 nM) was included as a positive control (a, d, c). Cell lysates were analyzed for the feedback activation of the PI-3 kinase pathway (a, b) or for LC3-II lipidation (c, d) by western blot with the indicated antibodies. g, h The effect of bafilomycin and colchicine on LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus bafilomycin (100 nM) (g, j) or colchicine (5 μM) (h, j) for 16 h. Cell lysates were analyzed for LC3 and actin expression by western blot. i, j Inability of uridine to block A77 1726-induced LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus uridine (200 μM) for 16 h. Cell lysates were analyzed for LC3-II lipidation and actin expression by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, f, j). LC3 lipidation was analyzed by comparing the density of LC3-II with β-actin. The data in Fig. 1c, f, j and the remaining Image-J-derived data in all other figures are the mean ± SD from three experiments. k, l NSC34 cells were transfected with the expression vector pmLC3-RFP. The cells were left untreated or treated with A77 1726 (200 μM) or rapamycin (50 nM) for 16 h. Autophagosomes were visualized under a confocal microscope (k). The puncta of autophagosomes were counted under a fluorescent microscope and plotted in a bar graph with statistical analysis (l). *p < 0.05; **p < 0.01. m Schematic model of A77 1726-induced autophagy. Inhibition of S6K1 activity leads to TAK1 activation, which activates AMPK. AMPK phosphorylates ULK1S555 and activates it. Inhibition of S6K1 by A77 1726 leads to the feedback activation of the PI-3 kinase pathway, as evidenced by increased AKT and S6K1 phosphorylation. Rapamycin also induces the feedback activation of the PI-3 kinase pathway. However, rapamycin targets mTOR, leading to decreased S6K1 phosphorylation
The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and autophagy.
a–f The effect of A77 1726 on the feedback activation of the PI-3 kinase pathway and LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 h (a, c, d) or were incubated in the presence of A77 1726 (200 μM) for the indicated time (b, e, f). Rapamycin (50 nM) was included as a positive control (a, d, c). Cell lysates were analyzed for the feedback activation of the PI-3 kinase pathway (a, b) or for LC3-II lipidation (c, d) by western blot with the indicated antibodies. g, h The effect of bafilomycin and colchicine on LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus bafilomycin (100 nM) (g, j) or colchicine (5 μM) (h, j) for 16 h. Cell lysates were analyzed for LC3 and actin expression by western blot. i, j Inability of uridine to block A77 1726-induced LC3-II lipidation. NSC34 cells were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) minus or plus uridine (200 μM) for 16 h. Cell lysates were analyzed for LC3-II lipidation and actin expression by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, f, j). LC3 lipidation was analyzed by comparing the density of LC3-II with β-actin. The data in Fig. 1c, f, j and the remaining Image-J-derived data in all other figures are the mean ± SD from three experiments. k, l NSC34 cells were transfected with the expression vector pmLC3-RFP. The cells were left untreated or treated with A77 1726 (200 μM) or rapamycin (50 nM) for 16 h. Autophagosomes were visualized under a confocal microscope (k). The puncta of autophagosomes were counted under a fluorescent microscope and plotted in a bar graph with statistical analysis (l). *p < 0.05; **p < 0.01. m Schematic model of A77 1726-induced autophagy. Inhibition of S6K1 activity leads to TAK1 activation, which activates AMPK. AMPK phosphorylates ULK1S555 and activates it. Inhibition of S6K1 by A77 1726 leads to the feedback activation of the PI-3 kinase pathway, as evidenced by increased AKT and S6K1 phosphorylation. Rapamycin also induces the feedback activation of the PI-3 kinase pathway. However, rapamycin targets mTOR, leading to decreased S6K1 phosphorylationTAK1 is a serine/threonine kinase activated by IL-1 and TGF-β receptors, Toll-like receptors, CD40, and B cell receptor[17-19]. TAK1 plays important roles in cell survival, differentiation, apoptosis, and inflammatory responses. Recent studies have shown that TAK1 inactivation mutations cause frontometaphyseal dysplasia[20] and cardiospondylocarpofacial syndrome[21]. TAK1 phosphorylates and activates several intracellular kinases, including p38, c-Jun N-terminal kinase (JNK), and I-kappa B kinase complex (IKK)[22-25]. In addition, TAK1 also activates the tumor suppressor protein LKB1, leading to AMPKT172 phosphorylation and activation (Fig. 1m)[26]. Inokuchi-Shimizu et al[27]. reported that TAK1 is required for starvation-induced AMPK and ULK1 phosphorylation and activation, and plays a critical role in inducing autophagy. Moreover, TAK1 deficiency partially blocks rapamycin-induced autophagy in hepatocytes[27]. Mechanisms by which TAK1 promotes autophagy and its involvement in clearing protein aggregates remain to be defined.Leflunomide (AravaTM) is an anti-inflammatory drug approved for treating rheumatoid arthritis (RA). A77 1726 and its parental drug, leflunomide, inhibit tyrosine phosphorylation and pyrimidine nucleotide synthesis[28-35]. The ability of A77 1726 to inhibit the activity of dihydroorotate dehydrogenase (DHO-DHase), a rate-limiting enzyme in pyrimidine nucleotide synthesis, is much stronger than its ability to inhibit the activity of protein tyrosine kinases such as p56lck, p59fyn, and platelet-derived growth factor (PDGF) receptor[28-32]. Our recent study showed that leflunomide and its active metabolite A77 1726 directly inhibit the activity of purified p70 S6 kinase (S6K1) in an in vitro kinase assay. Inhibition of S6K1 in an A375melanoma cell line by A77 1726 leads to the feedback activation of the PI-3 kinase pathway as evidenced by increased AKT and S6K1 phosphorylation but modestly or weakly decreased S6 phosphorylation (Fig. 1m)[36]. Here we report that A77 1726 induced autophagy and SOD1 degradation in NSC34 cells through TAK1-induced AMPK activation (Fig. 1m).
Results
Autophagy induction by A177 1726 in NSC34 cells
Consistent with our prior observations[36], A77 1726 increased AKTS473 and S6K1T389 phosphorylation in a dose-dependent manner in NSC34 cells (Fig. 1a,c). A77 1726 rapidly induced AKTS473 and S6K1T389 phosphorylation, as soon as 2 h after exposure to A77 1726 (Fig. 1b,f). A77 1726 modestly inhibited S6 phosphorylation due to S6K1 hyperactivation (Fig. 1a, b), a phenomenon consistent with the observation made with other S6K1 inhibitors such as PF-4708671 as shown below (Fig. 2b). We next tested if mTOR feedback activation by A77 1726 led to the inhibition of autophagy. Surprisingly, A77 1726 increased LC3-II lipidation in a dose- (Fig. 1d) and time-dependent (Fig. 1e) manner in NSC34 cells. Rapamycin included as a positive control modestly increased LC3-II levels (Fig. 1d). Increased LC3-II lipidation was not due to the stall of autophagy flux since combination of A77 1726 with bafilomycin (Fig. 1g,j) or colchicine (Fig. 1h,j) increased the levels of LC3-II and increased the ratios of LC3-II to LC-I, compared to bafilomycin or colchicine alone. A77 1726 inhibits pyrimidine nucleotide synthesis by inhibiting DHO-DHase activity[28, 29]. Uridine can be used to normalize pyrimidine nucleotide levels in vitro and in vivo[28, 29]. We found that exogenous uridine (200 μM) was unable to block A77 1726-induced increase of LC3-II levels (Fig. 1i, j), suggesting that increased LC3 lipidation by A77 1726 was not due to its inhibitory effect on pyrimidine nucleotide synthesis. Confocal microscopic fluorescence analysis revealed that LC3 formed autophagosomes in NSC34 cells in the presence of A77 1726 or rapamycin (Fig. 1k). Statistical analysis revealed that the number of autophagosome puncta was significantly higher in NSC34 cells treated with A77 1726 or rapamycin than that in the untreated controls (Fig. 1l).
Fig. 2
S6K1 inhibition induces autophagy.
a, c The effect of S6K1 knockdown on LC3-II lipidation. NSC34 cells were transfected with scrambled or S6K1 siRNA (2.5 nmole each). After incubation for 48 h, cell lysates were prepared and analyzed for total and phosphorylated proteins by western blot. b, d The effect of the S6K1 inhibitor on LC3-II expression. NSC34 cells seeded in 6-well plates were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of PF-4708671 for 16 h. Cell lysates were analyzed for total and phosphorylated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, d). e The effect of S6K1 knockdown on autophagosome formation. NSC34 cells seeded on the coverslips were first transfected with scrambled or S6K1 siRNA (2.5 nmole each). After incubation overnight, the cells were transfected with pmLC3-RFP expression vector. After incubation for 48 h, the cells were fixed in methanol and visualized for autophagosomes under a confocal fluorescent microscope. f The effect of the S6K1 inhibitor on LC3-II expression. NSC34 cells seeded on coverslips were transfected with LC3-RFP expression vector. After incubation for 24 h, the cells were treated with DMSO (0.2%) or PF-4708671 (20 μM) for 16 h. Cells were fixed and analyzed for autophagosomes under a fluorescent microscope. g, h The puncta of autophagosomes were counted under a fluorescent microscope and plotted in a bar graph with statistical analysis. *p < 0.05; **p < 0.01
S6K1 inhibition induces autophagy.
a, c The effect of S6K1 knockdown on LC3-II lipidation. NSC34 cells were transfected with scrambled or S6K1 siRNA (2.5 nmole each). After incubation for 48 h, cell lysates were prepared and analyzed for total and phosphorylated proteins by western blot. b, d The effect of the S6K1 inhibitor on LC3-II expression. NSC34 cells seeded in 6-well plates were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of PF-4708671 for 16 h. Cell lysates were analyzed for total and phosphorylated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, d). e The effect of S6K1 knockdown on autophagosome formation. NSC34 cells seeded on the coverslips were first transfected with scrambled or S6K1 siRNA (2.5 nmole each). After incubation overnight, the cells were transfected with pmLC3-RFPexpression vector. After incubation for 48 h, the cells were fixed in methanol and visualized for autophagosomes under a confocal fluorescent microscope. f The effect of the S6K1 inhibitor on LC3-II expression. NSC34 cells seeded on coverslips were transfected with LC3-RFPexpression vector. After incubation for 24 h, the cells were treated with DMSO (0.2%) or PF-4708671 (20 μM) for 16 h. Cells were fixed and analyzed for autophagosomes under a fluorescent microscope. g, h The puncta of autophagosomes were counted under a fluorescent microscope and plotted in a bar graph with statistical analysis. *p < 0.05; **p < 0.01
Autophagy induction by suppression of S6K1 expression or activity
We next tested if S6K1 siRNA also induced autophagy in NSC34 cells. As shown in Fig. 2a, c, S6K1 siRNA reduced S6K1expression and S6 phosphorylation but increased AKT phosphorylation and LC3-II lipidation. PF-4708671, a specific inhibitor of S6K1, modestly inhibited S6 phosphorylation but induced the feedback activation of the PI-3 kinase pathway, evidenced by increased AKT and S6K1 phosphorylation (Fig. 2b, d). Consistently, PF-4708671 increased the ratio of LC3-II/LC3-I in a dose-dependent manner in NSC34 cells (Fig. 2b, d). Both S6K1 knockdown and PF-4708671 increased the number of LC3-RFP puncta (Fig. 2e, f) in NSC34 cells. The number of autophagosome puncta was significantly higher in NSC34 cells with S6K1 knockdown or treated with PF-4708671 than their corresponding controls (Fig. 2g, h).
Inhibition of S6K1 activity leads to AMPK and ULK1 phosphorylation
AMPK phosphorylates ULK1S555 and induces autophagy[12, 37]. Here we tested if A77 1726 induced autophagy by phosphorylating and activating AMPK and ULK1. A77 1726 significantly increased AMPKT172, ULK1S555, and acetyl-CoA carboxylase (ACCS79) (another substrate of AMPK) phosphorylation in NSC34 cells even at 50 μM (Fig. 3a, c) and in a time-dependent (Fig. 3b, d) manner. mTOR is activated by A77 1726 due to the feedback activation of the PI-3 kinase pathway[36]. A77 1726 induced ULK1S757 phosphorylation in a dose- and time-dependent manner (Fig. 3a–d). Rapamycin did not increase AMPKT172 and ULK1S555 phosphorylation but suppressed ULK1S757 phosphorylation (Fig. 3a,c). Consistent with these observations, suppression of S6K1expression by S6K1 siRNA (Fig. 3e, g) or inhibition of S6K1 activity by PF-4708671 (Fig. 3f, h) led to increased AMPKT172, ULK1S555, ULK1S757, and ACCS79 phosphorylation.
Fig. 3
AMPK and ULK1 phosphorylation by S6K1 inhibition.
NSC34 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 h (a, c) or in the presence of A77 1726 (200 μM) for the indicated time (b, d). NSC34 cells were transfected with S6K1 siRNA and incubated for 48 h (e, g) or were treated with the indicated concentrations of PF-4708671 for 16 h (f, h). Cell lysates were analyzed for total and phosphorylated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, d, g, h). *p < 0.05; **p < 0.01, compared to the untreated control
AMPK and ULK1 phosphorylation by S6K1 inhibition.
NSC34 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 h (a, c) or in the presence of A77 1726 (200 μM) for the indicated time (b, d). NSC34 cells were transfected with S6K1 siRNA and incubated for 48 h (e, g) or were treated with the indicated concentrations of PF-4708671 for 16 h (f, h). Cell lysates were analyzed for total and phosphorylated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, d, g, h). *p < 0.05; **p < 0.01, compared to the untreated control
Evidence that AMPK mediates A77 1726-induced ULK1 phosphorylation and autophagy
We then determined if AMPK activation by A77 1726 was indeed responsible for ULK1 phosphorylation and autophagy. As shown in Fig. 4a, b, two AMPK activators, oligomycin and metformin, induced AMPKT172 and ULK1S555 phosphorylation but had no effect on ULK1S757 phosphorylation. Oligomycin was more potent than metformin in inducing LC3-II lipidation. Compound C (CC), an inhibitor of AMPK, did not significantly inhibit A77 1726-induced ULK1S757 phosphorylation but blocked A77 1726-induced AMPKT172 and ULK1S555 phosphorylation as well as LC3-II lipidation (Fig. 4c, d).
Fig. 4
Role of AMPK in A77 1726-induced autophagy.
NSC34 cells were incubated in the absence or presence of A77 1726 (200 μM), oligomycin (5 μM) or metformin (10 mM) for 16 h (a, b) or were incubated in the absence or presence of A77 1726 (200 μM) and/or compound C (5 μM) for 16 h (c, d). Cell lysates were prepared and analyzed for total and phosphorylated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (b, d). *p < 0.05; **p < 0.01
Role of AMPK in A77 1726-induced autophagy.
NSC34 cells were incubated in the absence or presence of A77 1726 (200 μM), oligomycin (5 μM) or metformin (10 mM) for 16 h (a, b) or were incubated in the absence or presence of A77 1726 (200 μM) and/or compound C (5 μM) for 16 h (c, d). Cell lysates were prepared and analyzed for total and phosphorylated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (b, d). *p < 0.05; **p < 0.01
Role of TAK1 in S6K1-mediated regulation of autophagy
We next tested if S6K1 suppression by A77 1726 led to the activation of TAK1, subsequently activating AMPK. As shown in Fig. 5a, b, 5Z-7-oxozeaenol, an inhibitor of TAK1, blocked A77 1726-induced phosphorylation of TAK1T184/187, AMPKT172, and ULK1S555, and blocked A77 1726-induced LC3-II lipidation. TAK1 siRNA suppressed TAK1expression (Fig. 5c, d). Consistently, suppression of TAK1 by siRNA led to the inhibition of A77 1726-induced phosphorylation of AMPKT172, ULK1S555, and TAK1T184/187, and blocked A77 1726-induced LC3-II lipidation (Fig. 5c, d). Both 5Z-7-oxozeaenol and S6K1 siRNA somehow also partially blocked A77 1726-induced ULK1S757 phosphorylation.
Fig. 5
TAK1 mediates A77 1726-induced AMPK activation.
NSC34 cells were treated with A77 1726 (200 μM) and/or 5Z-7-oxozeaenol (100 nM) for 16 h (a, b) or were transfected with scrambled or TAK1 siRNA (2.5 nmole each) (c, d). After incubation for 48 h, the cells were left untreated or treated with A77 1726 for 16 h. Cell lysates were prepared and analyzed for the expression of the indicated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, d). *p < 0.05; **p < 0.01
TAK1 mediates A77 1726-induced AMPK activation.
NSC34 cells were treated with A77 1726 (200 μM) and/or 5Z-7-oxozeaenol (100 nM) for 16 h (a, b) or were transfected with scrambled or TAK1 siRNA (2.5 nmole each) (c, d). After incubation for 48 h, the cells were left untreated or treated with A77 1726 for 16 h. Cell lysates were prepared and analyzed for the expression of the indicated proteins by western blot. The expression levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs (c, d). *p < 0.05; **p < 0.01
Autophagy plays a critical role in A77 1726-induced SOD1 degradation
A77 1726 slightly reduced the number of wild-type SOD1-GFP-positive NSC34 cells but modestly reduced the number of SOD1G93A-GFP-positive NSC34 cells (Fig. 6a, b). In contrast, rapamycin had little effect in the number of both wild-type SOD1-GFP and mutant SOD1G93A-GFP-positive NSC34 cells (Fig. 6a, b). To quantify the reduction of GFP-positive cells, NSC34 cells transfected with wild-type SOD1-GFP or mutant SOD1G93A-GFP expression vector in the absence or presence of A77 1726 or rapamycin were analyzed for the fluorescent intensity of the GFP-positive cells in a plate reader. As shown in Fig. 6c, the fluorescence intensity was significantly reduced in A77 1726-treated NSC34 cells transfected with either wild-type SOD1-GFP or SOD1G93A-GFP expression vector, compared to that in untreated controls. The fluorescence intensity was slightly reduced in rapamycin-treated NSC34 cells, but that was not statistically significant. Of note, the decreased fluorescence intensity in A77 1726-treated cells was not caused by A77 1726-mediated anti-proliferative effect since the GFP fluorescence intensity was normalized against the fluorescence intensity from the nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI).
Fig. 6
A77 1726 blocks the formation of SOD1G93A protein aggregates.
a, b NSC34 cells transiently transfected with SOD1-GFP or SOD1G93A-GFP expression vectors were treated as described in “Materials and Methods” section. The cells were examined under a confocal microscope for SOD1-GFP or SOD1G93A-GFP expression (a, b) and quantified for the fluorescence intensity in a plate reader (c). The results represent the mean ± SD from one experiment in triplicate. The experiments were repeated twice with similar results. d The anti-proliferative effect of A77 1726 on NSC34 cells. Untransfected NSC34 cells or the cells transfected with the SOD1-GFP or SOD1G93A-GFP expression vector were seeded in a 96-well plate (5000 cells per well) and incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (50 nM) for 24 h. Cell proliferation was analyzed by an ATP-based Cell-Glo assay. The data are the mean ± SD of the triplicate from one representative of two experiments with similar results. *p < 0.05; **p < 0.01
A77 1726 blocks the formation of SOD1G93A protein aggregates.
a, b NSC34 cells transiently transfected with SOD1-GFP or SOD1G93A-GFP expression vectors were treated as described in “Materials and Methods” section. The cells were examined under a confocal microscope for SOD1-GFP or SOD1G93A-GFP expression (a, b) and quantified for the fluorescence intensity in a plate reader (c). The results represent the mean ± SD from one experiment in triplicate. The experiments were repeated twice with similar results. d The anti-proliferative effect of A77 1726 on NSC34 cells. Untransfected NSC34 cells or the cells transfected with the SOD1-GFP or SOD1G93A-GFP expression vector were seeded in a 96-well plate (5000 cells per well) and incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (50 nM) for 24 h. Cell proliferation was analyzed by an ATP-based Cell-Glo assay. The data are the mean ± SD of the triplicate from one representative of two experiments with similar results. *p < 0.05; **p < 0.01To rule out the possibility that A77 1726 reduced SOD1-GFP fluorescence intensity by selectively killing NSC34 cells expressing SOD1-GFP protein, we measured the proliferation index of untransfected NSC34 cells or NSC34 cells transfected with SOD1-GFP or SOD1G93A-GFP expression vector in the absence or presence of A77 1726 (200 μM) or rapamycin (50 nM). As shown in Fig. 6d, A77 1726 inhibited the proliferation of untransfected NSC34 cells slightly better than the NSC34 cells transfected with the SOD1-GFP or SOD1G93A-GFP expression vector. Rapamycin did not significantly inhibit NSC34 cell proliferation.Flow cytometry revealed that A77 1726 significantly shifted the peak of SOD1-GFP- and SOD1G93A-GFP-transfected cells to the left side; whereas rapamycin had little effect in shifting the peaks of GFP-positive cells (Fig. 7a). Western blot revealed that a very light smear of SOD1 protein aggregates was seen in wild-type SOD1-transfected NSC34 cells (Fig. 7b). In contrast, there was a very heavy smear of SOD1 mutant proteins in the insoluble fractions of GFP-SOD1G93A-transfected NSC34 cells (Fig. 7b). Of note, GFP-SOD1 monomer marked in Fig. 7b was detected as a heavy band with a molecular weight of ~53 kDa protein, whereas protein aggregates were detected as a dimer of ~100 kDa or multimer with heavier molecular weights. A77 1726 did not reduce wild-type SOD1 protein aggregates but reduced the smear of mutant SOD1 protein aggregates in NSC34 cells (Fig. 7b, d). Rapamycin did not significantly reduce the light smear of wild-type SOD1 aggregates nor reduced the heavy smear of mutant SOD1 aggregates in NSC34 cells (Fig. 7b, d). A77 1726 did not reduce the light smear of wild-type SOD1 protein aggregates but significantly reduced mutant SOD1 aggregates in a dose-dependent manner (Fig. 7c, e).
Fig. 7
Evidence that A77 1726 induces SOD1 protein degradation.
a NSC34 cells seeded in 60 mm dishes were transfected the SOD1-GFP or SOD1G93A-GFP expression vectors and treated with A77 1726 (200 μM) or rapamycin (50 nM). Single-cell suspensions were analyzed for GFP expression in a flow cytometer. The fluorescence intensity was analyzed by using FlowJo software. The results represent the mean ± SD from three independent experiments. *p < 0.05; **p < 0.01. b, c Western blot analysis of SOD1 aggregates. SOD1-GFP or SOD1G93A-GFP-transfected NSC34 cells were treated with A77 1726 or rapamycin as above (b) or treated with the indicated concentrations of A77 1726 (c) for 24 h. Insoluble fractions of the cell lysates were analyzed by western blot with an anti-SOD1 rabbit serum or actin. Protein aggregates as marked were analyzed by using a NIH Image J software and presented as bar graphs (d, e). *p < 0.05; **p < 0.01
Evidence that A77 1726 induces SOD1 protein degradation.
a NSC34 cells seeded in 60 mm dishes were transfected the SOD1-GFP or SOD1G93A-GFP expression vectors and treated with A77 1726 (200 μM) or rapamycin (50 nM). Single-cell suspensions were analyzed for GFP expression in a flow cytometer. The fluorescence intensity was analyzed by using FlowJo software. The results represent the mean ± SD from three independent experiments. *p < 0.05; **p < 0.01. b, c Western blot analysis of SOD1 aggregates. SOD1-GFP or SOD1G93A-GFP-transfected NSC34 cells were treated with A77 1726 or rapamycin as above (b) or treated with the indicated concentrations of A77 1726 (c) for 24 h. Insoluble fractions of the cell lysates were analyzed by western blot with an anti-SOD1rabbit serum or actin. Protein aggregates as marked were analyzed by using a NIH Image J software and presented as bar graphs (d, e). *p < 0.05; **p < 0.01Confocal microscopy revealed that both A77 1726 and rapamycin induced the formation of autophagosomes in wild-type SOD1-GFP- and mutant SOD1G93A-GFP-transfected NSC34 cells (Fig. 8a). RFP-LC3 autophagosomes were not co-localized with wild-type SOD1-GFP proteins in NSC34 cells in the absence or presence of A77 1726 or rapamycin. In contrast, RFP-LC3 autophagosomes were precisely co-localized with mutant SOD1G93A-GFP aggregates in A77 1726-treated NSC34 cells. In contrast, rapamycin induced relatively poor co-localization of RFP-LC3 autophagosomes with mutant SOD1G93A-GFP aggregates in NSC34 cells (Fig. 8b).
Fig. 8
A77 1726 induces SOD1G93A co-localization with autophagosomes.
RFP-LC3 stably transfected NSC34 cells were transiently transfected with SOD1-GFP (a) or SOD1G93A-GFP (b) expression vectors. After incubation for 40 h, the cells were treated with DMSO (0.2%), A77 1726 (200 μM) or rapamycin (50 nM) (a) for 24 h. The cells were fixed and examined under a confocal microscope for the localization of autophagosomes (RFP-LC3) and for SOD1-GFP or SOD1G93A-GFP protein aggregates. c–e NSC34 cells were transfected with control or ATG7 siRNA (100 nmole each) and with SOD1-GFP or SOD1G93A-GFP expression vectors. After incubation for 48 h, the cells were collected. Cell lysates were loaded to a non-reducing gel followed by western blot analysis with indicated antibodies. The data in Fig. 8d were derived from Fig. 8c in which only the density of protein aggregates (excluding the heavy band of the 53 kDa monomer) was quantified. Data in Fig. 8e were derived from Fig. 8c in which the relative levels of ATG7 and LC3 lipidation were analyzed. *p < 0.05; **p < 0.01
A77 1726 induces SOD1G93A co-localization with autophagosomes.
RFP-LC3 stably transfected NSC34 cells were transiently transfected with SOD1-GFP (a) or SOD1G93A-GFP (b) expression vectors. After incubation for 40 h, the cells were treated with DMSO (0.2%), A77 1726 (200 μM) or rapamycin (50 nM) (a) for 24 h. The cells were fixed and examined under a confocal microscope for the localization of autophagosomes (RFP-LC3) and for SOD1-GFP or SOD1G93A-GFP protein aggregates. c–e NSC34 cells were transfected with control or ATG7 siRNA (100 nmole each) and with SOD1-GFP or SOD1G93A-GFP expression vectors. After incubation for 48 h, the cells were collected. Cell lysates were loaded to a non-reducing gel followed by western blot analysis with indicated antibodies. The data in Fig. 8d were derived from Fig. 8c in which only the density of protein aggregates (excluding the heavy band of the 53 kDa monomer) was quantified. Data in Fig. 8e were derived from Fig. 8c in which the relative levels of ATG7 and LC3 lipidation were analyzed. *p < 0.05; **p < 0.01ATG7 siRNA reduced ATG7expression by ~30% in both wild-type SOD1-GFP- and SOD1G93A-GFP-transfected cells (Fig. 8c, e). Suppression of ATG7expression by ATG7 siRNA also blocked A77 1726-induced LC3 lipidation in NSC34 cells transfected with the wild-type SOD1-GFP- or SOD1G93A-GFP expression vector (Fig. 8c,e). ATG7 siRNA had no effect on SOD1-GFP expression levels in untreated NSC34 cells. A77 1726 had little effect on the levels of wild-type SOD1-GFP aggregates. However, A77 1726 significantly reduced the amount of SOD1G93A-GFP, which was blocked by ATG7 siRNA (Fig. 8c, d). Of note, the data in Fig. 8d were derived from Fig. 8c, in which only the density of protein aggregates (excluding the heavy band of the 53-kDa monomer) was quantified.
Discussion
It is well established that mTOR phosphorylates ULK1S757, inhibits ULK1 activity, and suppresses autophagy. Inhibition of mTOR activity by rapamycin or nutrient starvation leads to ULK1 activation and autophagy[38, 39]. In the present study, we demonstrated that mTOR feedback activation by two S6K1 inhibitors, A77 1726 or PF-4708671, and by S6K1 siRNA did not suppress autophagy in a motoneuron cell line. Instead, S6K1 inhibitors and S6K1 siRNA induced autophagy. It appears that, even though ULK1 is phosphorylated at S757, it remains to be active in A77 1726-treated NSC34 cells, probably due to its phosphorylation at S555. Loss of function of Tuberous Sclerosis Complex 1 (TSC1) or TSC2 in the setting of the genetic condition, Tuberous Sclerosis Complex, activates mTORC1 and downregulates the basal level autophagy in dividing cells[40]. Interestingly, TSC2-deficient neurons with heightened mTOR activity have an efficient autophagic process through compensatory AMPK activation and increased ULK1S555 phosphorylation[40].We and others have recently demonstrated that A77 1726 and leflunomide induce autophagy in renal carcinoma and melanoma cell lines[41, 42]. Our present study focuses on the mechanisms of A77 1726-induced autophagy and its impact on degrading misfolded protein aggregates in a motor neuron cell line. A77 1726 has three biochemical activities: inhibition of pyrimidine nucleotide synthesis by inhibiting DHO-DHase activity, inhibition of PTK activities, and inhibition of S6K1 activity. Exogenous uridine was unable to block A77 1726-induced autophagy, suggesting that A77 1726-induced autophagy is independent of its inhibitory effect on pyrimidine nucleotide synthesis. Of note, the concentration of uridine used in our study was 200 μM. This uridine concentration or even lower, which has been widely reported in literature by others[43] or by ourselves[28, 30], is sufficient to normalize intracellular pyrimidine levels in cells treated with A77 1726 or other more potent DHO-DHase inhibitors such as brequinar sodium. Furthermore, inhibition of S6K1 activity by PF-4708671 or S6K1 siRNA included as controls also induced autophagy. A77 1726-induced autophagy is likely mediated by its inhibition of S6K1 activity. Consistent with this notion, Park et al.[44] recently reported that PF-4708671 induces autophagy in mouse embryonic fibroblasts and promotes p62-dependent autophagic degradation of Keap1 protein.Based on the observations that inhibition of TAK1 activity by a specific inhibitor 5Z-7-oxozeaenol and by TAK1 siRNA blocked A77 1726-induced LC3-II lipidation, we postulate that TAK1 is responsible for S6K1 inhibition-induced autophagy (Fig. 1m). In support of this notion, Kim et al.[45] reported that S6K1 negatively regulates the activity of TAK1. Inokuchi-Shimizu et al.[27] showed that TAK1 deficiency leads to the inhibition of starvation-induced autophagy in the liver of TAK1 knockout mice. These investigators further showed that TAK1 deficiency compromises rapamycin-induced autophagy in the hepatocytes of TAK1 knockout mice. These observations collectively suggest that TAK1 plays a key role in mediating the S6K1 inhibitor-induced autophagy (Fig. 1m).Several studies suggest that TAK1 induces autophagy through AMPK activation. TAK1 activates AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells[46]. TAK1 is responsible for VEGF–induced AMPK activation in endothelial cells[47]. AMPK phosphorylation at T172 and its activity are subdued in TAK1-null embryos[26]. Consistent with these observations, our present study showed that TAK1 siRNA and 5Z-7-oxozeaenol blocked A77 1726-induced AMPK activation. The AMPK inhibitor compound C blocked A77 1726-induced ULK1S555 phosphorylation and autophagy. These observations collectively suggest that TAK1 plays a critical role in A77 1726-induced AMPK activation.Previous studies have shown that S6K1 deficiency leads to AMPK activation in the skeletal muscle tissues and myotubes of S6K1-deficientmice due to increased AMP levels and AMP/ATP ratios[48, 49]. It remains to be determined if A77 1726-induced AMPK activation is in part mediated by increased AMP levels and AMP/ATP ratio. A recent study showed that Fyntyrosine kinase phosphorylates AMPK at Y436 and suppresses its activation, as evidenced by decreased AMPK phosphorylation at T172 in TNF-α-treated HEK293 cells[50]. Our early study showed that A77 1726 is an inhibitor of the Src family tyrosine kinases p56Lck and p59Fyn[31]. A77 1726 seemed to induce AMPKT172 and ULK1S555 phosphorylation at a lower concentration (Fig. 3a) than that required for inhibition of S6K1 activity (Fig. 1a). In addition, though PF-4708671 is more potent at inhibiting S6K1 activity than A77 1726, PF-4708671 was less effective at inducing LC3-II lipidation (Fig. 2b) and ULK1S555 phosphorylation than did A77 1726 in NSC34 cells. It is possible that A77 1726 may also activate AMPK and induce autophagy by inhibiting Fyntyrosine kinase activity. Moreover, S6K1 binds to and phosphorylates AMPK α2 at S491, and inhibits AMPK activity[51]. S6K1 may regulate AMPK activity by multiple mechanisms.In the present study, we found that A77 1726 induced autophagy and mutant SOD1G93A degradation in NSC34 cells. Mutant SOD1G93A aggregates were co-localized with autophagosomes. In contrast, rapamycin, though it also induced the formation of autophagosomes, had limited effect on inducing mutant SOD1 degradation as evidenced by minimal reduction of protein aggregates in western blot and fluorescent microscopic analysis. Moreover, LC3-RFP autophagosomes did not precisely co-localize with mutant SOD1 aggregates in rapamycin-treated cells. A77 1726 appears to be more effective than rapamycin in inducing SOD1 degradation. We speculate that autophagy induced by A77 1726 through AMPK activation is more robust than rapamycin-induced autophagy in motor neurons with misfolded protein aggregates. Saxena et al. reported that mTOR activation protects ALS motoneurons, delays ALS onset, and extends survival[52]. Lithium and trehalose, two AMPK activators, provide neuroprotective effects, delay ALS onset, and prolong survival in animal models[53-56]. mTOR feedback activation by S6K1 inhibitors may protect motor neurons from apoptosis. Indeed, motoneuron apoptosis is exacerbated in rapamycin-treated SOD1G93A-transgenic mice[57]. Recent studies showed that Src/c-Abltyrosine kinases are highly activated in the motor neurons of ALSpatients[58, 59]. Inhibition of Srcexpression by siRNA and activity by the Src inhibitor bosutinib induces autophagy and increases the survival of motor neurons derived from patients with SOD1G93A gene mutation[58]. Our prior studies have shown that A77 1726 also inhibits the activity of the Src family tyrosine kinases p56Lck and p59Fyn[31]. Leflunomide may function as a potent autophagy activator by targeting multiple molecules.Leflunomide is a novel disease-modifying anti-RA drug. Its active metabolite, A77 1726, inhibits S6K1 activity with the IC50 values of ~50–75 μM[36]. Plasma concentrations of A77 1726 in RApatients treated with leflunomide (20 mg/day) are higher than 200 μM[60]. A77 1726 in the blood of mice treated with leflunomide at a dose of 35 mg/kg has a remarkably long half-life of 15 h. The blood concentrations of A77 1726 reached a peak of 500 μM within 4 h and remained at 250 μM at 24 h after a single dose of 35 mg/kg of leflunomide in mice[61]. Our present study showed that A77 1726 concentrations between 50 and 200 μM were very effective in inducing SOD1 mutant protein degradation (Fig. 6) and autophagy (Fig. 1). These observations suggest that the concentrations of A77 1726 used in our study are physiologically relevant. Rapamycin induced autophagy at the concentrations of nanomolar ranges, which are much lower than A77 1726 required to induce autophagy. It should be noted that the IC50 value required for rapamycin to inhibit its molecular target, mTOR, is also dramatically lower than the IC50 value of A77 1726 required to inhibit its target, S6K1. Therefore, the low IC50 values for rapamycin to induce autophagy cannot be interpreted as being more potent in inducing autophagy than A77 1726 since rapamycin and leflunomide have totally different pharmacokinetics in vivo.In summary, our present study showed that inhibition of S6K1 activity by A77 1726 activates TAK1, leading to AMPK activation and autophagy (Fig. 1m). We further showed that A77 1726 induces SOD1 protein degradation in NSC34 cells through autophagy (Fig. 1m). Our study suggests that S6K1 can be targeted to induce autophagy, and that leflunomide may have potential to be used as a novel drug for treating ALS.
Materials and methods
Reagents
Leflunomide and A77 1726 were kindly provided by Cinkate Corporation (Oak Park, IL). SP600125 was purchased from Cell Signaling Technology (Danvers, MA). Rapamycin was purchased from Cayman Laboratories (Ann Arbor, MI). Bafilomycin, colchicine, metformin, 5Z-7-oxozeaenol, PF-4708671, and oligomycin were purchased from Sigma (St. Louis, MO). Anti-actin mAb was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against LC3, ULK1, AMPK, AKT, S6K1, S6, ACC (acetyl-CoA carboxylase) and their corresponding phospho-antibodies including ULK1S555, ULK1S757, AMPKT172, mTORS2448, AKTS473, S6K1T389, S6S235/236, ACCS79, and TAK1T184/187 were purchased from Cell Signaling Technology (Danvers, MA). Anti-SOD1 antibody was kindly provided by Dr. Han-Xiang Deng (Northwestern University, Chicago). The SOD1-GFP and SOD1G93A-GFP expression vectors were prepared by inserting a GFP gene downstream of SOD1 in a pcDNA3.1 vector. The expression vector encoding RFP-LC3 (pmRFP-LC3) was purchased from OriGene Technologies, Inc. (Rockville, MD). The NSC34 cell line was complete DMEM medium supplemented with 10% fetal bovine serum, streptomycin and penicillin, and L-glutamine.
Western blot
Cells grown in 6-well plates were collected and lysed in NP-40 lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). After incubation on ice for 30 min, the cell lysates were prepared by spinning down at 4 °C, 15,000 rpm for 15 min. For preparation of the fractions of soluble and insoluble proteins, NSC34 cells were lysed in extraction buffer (10 mM Tris-HCl pH 8.0, 1 mM EDTA, 100 mM NaCl, 0.5% NP-40, and a protease inhibitor cocktail 1:100 dilution (Thermo, Rockford, IL, USA)) followed by a brief sonication (50% output for 10 s with a probe sonicator (VCX 150, 150 W, Sonics, Newtown, CT, USA)). Cell lysates were spun down at 100,000 × g for 15 min at 4 °C. Pellets were resuspended in loading buffer (no β-mercapethanol) and followed by filtration through Qiagen DNA removal inserts to remove genomic DNA. Cell lysates were analyzed by western blot with antibodies against the proteins of interest, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG and SuperSignal Western Pico enhanced chemiluminoscence substrate (Pierce Chemical Co., Rockford, IL). The density of the bands was analyzed by using NIH Image-J software and normalized by the arbitrary units of their corresponding total proteins or β-actin as indicated. For analysis of LC3 lipidation, the lower band of LC3-II was used to compare with β-actin. All data derived from Image-J analyses were presented as the mean ± SD from three experiments in bar graphs.
S6K1, TAK1, and ATG7 knockdown
S6K1 siRNA ON-TARGETplus SMARTpool was synthesized by Dharmacon and purchased from Fisher Scientific (Pittsburg, PA). This S6K1 siRNA pool containing three different siRNAs has been previously shown to efficiently suppress S6K1expression[62, 63]. TAK1 and ATG7 siRNAs were purchased from Cell Signaling Technology (Danvers, MA). A scrambled control siRNA was purchased from Life Technologies (Invitrogen Life Technologies, Grand Island, NY). NSC34 cells seeded in 6-well plates were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies, Grand Island, NY) according to the manufacturer’s instruction. After incubation for 48 h, the cells were collected and analyzed for the expression of S6K1 and ATG7 and other relevant proteins by western blot. To determine the effect of ATG7 on A77 1726-induced SOD1 degradation, NSC34 cells were first transfected with control or ATG7 siRNA using Lipofectamine RNAiMAX, followed by transfection with SOD1-GFP or SOD1G93A-GFP expression vector. After incubation for 24 h, the cells were left untreated or treated with A77 1726 for 24 h. Insoluble fractions of cell lysates were prepared and analyzed for SOD1expression.
Fluorescent microscopy and flow cytometric analyses of SOD1 expression
NSC34 cells were transiently transfected with an expression vector encoding the wild-type or mutant SOD1G93A gene tagged with green fluorescence protein (GFP). Twenty-four hours later, SOD1-GFP and SOD1G93A-GFP-transfected cells were aliquoted into three wells in a 96-well plate. After incubation for 16 h, the cells were treated with dimethyl sulfoxide (DMSO) (0.2%), A77 1726 (200 μM) or rapamycin (50 nM) for 24 h. The cells were examined under a Nikon fluorescent microscope for SOD1-GFP or SOD1G93A-GFP expression. The cells were then fixed in methanol for 10 min at 4 °C. After air drying, the cells were replenished with 50 μl PBS per well. GFP fluorescence intensity was measured in a TECAN plate reader (Model Infinite M200 PRO) (Excitation 400 nm, Emission 508 nm). Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime Institute of Biotechnology Nantong, China). The plate was then read for DAPI fluorescence intensity with excitation and emission wavelengths of 359 and 461 nm, respectively. The relative GFP fluorescence intensity = (GFP reading in each well—the mean value of GFP readings from three untransfected wells)/(DAPI reading in each well—the mean value of three blank wells). For flow cytometric analysis of GFP-positive cells, NSC34 cells were similarly transfected, aliquoted into a 6-well plate, and treated with DMSO (0.2%), A77 1726 (200 μM) or rapamycin (50 nM) for 24 h as above. Single-cell suspensions were run in a Beckman Coulter flow cytometer (Model CyAn ADP). The fluorescence intensity was analyzed by using FlowJo software. The results from three independent experiments were statistically analyzed by using the unpaired Student’s t test.
Cell proliferation assay
NSC34 cells seeded in a 12-well plate (5000 cells per well) were left untransfected or transfected with the SOD1-GFP or SOD1G93A-GFP expression vector. After incubation for 24 h, the cells were aliquoted into a 96-well plate (5000 cells per well) and incubated overnight. The cells were then incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (50 nM). After incubation for 24 h, cell proliferation was analyzed by using an ATP-based Cell-Glo assay (Promegan, Madison, WI) following the manufacturer’s instruction.
Autophagosome analysis
NSC34 cells seeded on coverslips were transiently transfected with RFP-LC3expression plasmid DNA using FuGENE6 following the manufacturer’s protocol. After incubation for 48 h, the cells were incubated in the presence of A77 1726 (200 μM), rapamycin (50 nM), or PF-4708671 (20 μM). After incubation for 16 h, the cells were fixed in 100% methanol at −20 °C for 10 min. The coverslips were mounted with 50% glycerin in PBS containing DAPI (0.5 μg/ml). Autophagosomes were examined under a Leica LP8 confocal microscope. The autophagosome puncta was examined under a Nikon fluorescence microscope. To determine the effect of S6K1 knockdown on autophagosome formation, NSC34 cells were transfected with control or S6K1 siRNA as described above. After incubation for 24 h, the cells were transfected with RFP-LC3 plasmid DNA again. After incubation for another 48 h, the coverslips were collected, fixed, and mounted on slides and examined for RFP fluorescence under a fluorescent microscope. Autophagosome puncta in NSC34 cells treated with various drugs or siRNA transfection were counted in 30 randomly selected fields under a 40 × objective in a blinded fashion. Results represent the mean ± SD (standard deviation) from three independent experiments. To determine whether SOD1 was co-localized with autophagosome, NSC34 cells stably transfected with RFP-LC3 was transiently transfected with SOD1-GFP or SOD1G93A-GFP. Twenty-four hours after transfection, the cells were treated with A77 1726 (200 μM) or rapamycin (50 nM) and then fixed and analyzed under a confocal microscope.
Statistical analysis
An unpaired Student t test was used to analyze the differences in the number of puncta, the differences in the arbitrary number of western blot data from the Image J analysis, the difference in the relative light units and fluorescence intensity in NSC34 cells treated with various drugs. The data were presented as mean ± SD (western blot data, cell proliferation data, and fluorescence intensity data) or standard error of the mean (SEM) (puncta data). A p value of < 0.05 was considered statistically significant. All statistics was performed with SigmaPlot 11 software (Systat Software, Inc, San Jose, CA).
Authors: Yossi Dagon; Elizabeth Hur; Bin Zheng; Kerry Wellenstein; Lewis C Cantley; Barbara B Kahn Journal: Cell Metab Date: 2012-06-21 Impact factor: 27.287
Authors: X Xu; J Shen; J W Mall; J A Myers; W Huang; L Blinder; T J Saclarides; J W Williams; A S Chong Journal: Biochem Pharmacol Date: 1999-11-01 Impact factor: 5.858
Authors: Min Xie; Dou Zhang; Jason R B Dyck; Yi Li; Hui Zhang; Masae Morishima; Douglas L Mann; George E Taffet; Antonio Baldini; Dirar S Khoury; Michael D Schneider Journal: Proc Natl Acad Sci U S A Date: 2006-11-03 Impact factor: 11.205
Authors: K Shirakabe; K Yamaguchi; H Shibuya; K Irie; S Matsuda; T Moriguchi; Y Gotoh; K Matsumoto; E Nishida Journal: J Biol Chem Date: 1997-03-28 Impact factor: 5.157
Authors: H X Deng; A Hentati; J A Tainer; Z Iqbal; A Cayabyab; W Y Hung; E D Getzoff; P Hu; B Herzfeldt; R P Roos Journal: Science Date: 1993-08-20 Impact factor: 47.728
Authors: Victor Aguilar; Samira Alliouachene; Athanassia Sotiropoulos; Andrew Sobering; Yoni Athea; Fatima Djouadi; Sylvain Miraux; Eric Thiaudière; Marc Foretz; Benoit Viollet; Philippe Diolez; Jean Bastin; Paule Benit; Pierre Rustin; David Carling; Marco Sandri; Renée Ventura-Clapier; Mario Pende Journal: Cell Metab Date: 2007-06 Impact factor: 27.287
Authors: Daniel J Klionsky; Amal Kamal Abdel-Aziz; Sara Abdelfatah; Mahmoud Abdellatif; Asghar Abdoli; Steffen Abel; Hagai Abeliovich; Marie H Abildgaard; Yakubu Princely Abudu; Abraham Acevedo-Arozena; Iannis E Adamopoulos; Khosrow Adeli; Timon E Adolph; Annagrazia Adornetto; Elma Aflaki; Galila Agam; Anupam Agarwal; Bharat B Aggarwal; Maria Agnello; Patrizia Agostinis; Javed N Agrewala; Alexander Agrotis; Patricia V Aguilar; S Tariq Ahmad; Zubair M Ahmed; Ulises Ahumada-Castro; Sonja Aits; Shu Aizawa; Yunus Akkoc; Tonia Akoumianaki; Hafize Aysin Akpinar; Ahmed M Al-Abd; Lina Al-Akra; Abeer Al-Gharaibeh; Moulay A Alaoui-Jamali; Simon Alberti; Elísabet Alcocer-Gómez; Cristiano Alessandri; Muhammad Ali; M Abdul Alim Al-Bari; Saeb Aliwaini; Javad Alizadeh; Eugènia Almacellas; Alexandru Almasan; Alicia Alonso; Guillermo D Alonso; Nihal Altan-Bonnet; Dario C Altieri; Élida M C Álvarez; Sara Alves; Cristine Alves da Costa; Mazen M Alzaharna; Marialaura Amadio; Consuelo Amantini; Cristina Amaral; Susanna Ambrosio; Amal O Amer; Veena Ammanathan; Zhenyi An; Stig U Andersen; Shaida A Andrabi; Magaiver Andrade-Silva; Allen M Andres; Sabrina Angelini; David Ann; Uche C Anozie; Mohammad Y Ansari; Pedro Antas; Adam Antebi; Zuriñe Antón; Tahira Anwar; Lionel Apetoh; Nadezda Apostolova; Toshiyuki Araki; Yasuhiro Araki; Kohei Arasaki; Wagner L Araújo; Jun Araya; Catherine Arden; Maria-Angeles Arévalo; Sandro Arguelles; Esperanza Arias; Jyothi Arikkath; Hirokazu Arimoto; Aileen R Ariosa; Darius Armstrong-James; Laetitia Arnauné-Pelloquin; Angeles Aroca; Daniela S Arroyo; Ivica Arsov; Rubén Artero; Dalia Maria Lucia Asaro; Michael Aschner; Milad Ashrafizadeh; Osnat Ashur-Fabian; Atanas G Atanasov; Alicia K Au; Patrick Auberger; Holger W Auner; Laure Aurelian; Riccardo Autelli; Laura Avagliano; Yenniffer Ávalos; Sanja Aveic; Célia Alexandra Aveleira; Tamar Avin-Wittenberg; Yucel Aydin; Scott Ayton; Srinivas Ayyadevara; Maria Azzopardi; Misuzu Baba; Jonathan M Backer; Steven K Backues; Dong-Hun Bae; Ok-Nam Bae; Soo Han Bae; Eric H Baehrecke; Ahruem Baek; Seung-Hoon Baek; Sung Hee Baek; Giacinto Bagetta; Agnieszka Bagniewska-Zadworna; Hua Bai; Jie Bai; Xiyuan Bai; Yidong Bai; Nandadulal Bairagi; Shounak Baksi; Teresa Balbi; Cosima T Baldari; Walter Balduini; Andrea Ballabio; Maria Ballester; Salma Balazadeh; Rena Balzan; Rina Bandopadhyay; Sreeparna Banerjee; Sulagna Banerjee; Ágnes Bánréti; Yan Bao; Mauricio S Baptista; Alessandra Baracca; Cristiana Barbati; Ariadna Bargiela; Daniela Barilà; Peter G Barlow; Sami J Barmada; Esther Barreiro; George E Barreto; Jiri Bartek; Bonnie Bartel; Alberto Bartolome; Gaurav R Barve; Suresh H Basagoudanavar; Diane C Bassham; Robert C Bast; Alakananda Basu; Henri Batoko; Isabella Batten; Etienne E Baulieu; Bradley L Baumgarner; Jagadeesh Bayry; Rupert Beale; Isabelle Beau; Florian Beaumatin; Luiz R G Bechara; George R Beck; Michael F Beers; Jakob Begun; Christian Behrends; Georg M N Behrens; Roberto Bei; Eloy Bejarano; Shai Bel; Christian Behl; Amine Belaid; Naïma Belgareh-Touzé; Cristina Bellarosa; Francesca Belleudi; Melissa Belló Pérez; Raquel Bello-Morales; Jackeline Soares de Oliveira Beltran; Sebastián Beltran; Doris Mangiaracina Benbrook; Mykolas Bendorius; Bruno A Benitez; Irene Benito-Cuesta; Julien Bensalem; Martin W Berchtold; Sabina Berezowska; Daniele Bergamaschi; Matteo Bergami; Andreas Bergmann; Laura Berliocchi; Clarisse Berlioz-Torrent; Amélie Bernard; Lionel Berthoux; Cagri G Besirli; Sebastien Besteiro; Virginie M Betin; Rudi Beyaert; Jelena S Bezbradica; Kiran Bhaskar; Ingrid Bhatia-Kissova; Resham Bhattacharya; Sujoy Bhattacharya; Shalmoli Bhattacharyya; Md Shenuarin Bhuiyan; Sujit Kumar Bhutia; Lanrong Bi; Xiaolin Bi; Trevor J Biden; Krikor Bijian; Viktor A Billes; Nadine Binart; Claudia Bincoletto; Asa B Birgisdottir; Geir Bjorkoy; Gonzalo Blanco; Ana Blas-Garcia; Janusz Blasiak; Robert Blomgran; Klas Blomgren; Janice S Blum; Emilio Boada-Romero; Mirta Boban; Kathleen Boesze-Battaglia; Philippe Boeuf; Barry Boland; Pascale Bomont; Paolo Bonaldo; Srinivasa Reddy Bonam; Laura Bonfili; Juan S Bonifacino; Brian A Boone; Martin D Bootman; Matteo Bordi; Christoph Borner; Beat C Bornhauser; Gautam Borthakur; Jürgen Bosch; Santanu Bose; Luis M Botana; Juan Botas; Chantal M Boulanger; Michael E Boulton; Mathieu Bourdenx; Benjamin Bourgeois; Nollaig M Bourke; Guilhem Bousquet; Patricia Boya; Peter V Bozhkov; Luiz H M Bozi; Tolga O Bozkurt; Doug E Brackney; Christian H Brandts; Ralf J Braun; Gerhard H Braus; Roberto Bravo-Sagua; José M Bravo-San Pedro; Patrick Brest; Marie-Agnès Bringer; Alfredo Briones-Herrera; V Courtney Broaddus; Peter Brodersen; Jeffrey L Brodsky; Steven L Brody; Paola G Bronson; Jeff M Bronstein; Carolyn N Brown; Rhoderick E Brown; Patricia C Brum; John H Brumell; Nicola Brunetti-Pierri; Daniele Bruno; Robert J Bryson-Richardson; Cecilia Bucci; Carmen Buchrieser; Marta Bueno; Laura Elisa Buitrago-Molina; Simone Buraschi; Shilpa Buch; J Ross Buchan; Erin M Buckingham; Hikmet Budak; Mauricio Budini; Geert Bultynck; Florin Burada; Joseph R Burgoyne; M Isabel Burón; Victor Bustos; Sabrina Büttner; Elena Butturini; Aaron Byrd; Isabel Cabas; Sandra Cabrera-Benitez; Ken Cadwell; Jingjing Cai; Lu Cai; Qian Cai; Montserrat Cairó; Jose A Calbet; Guy A Caldwell; Kim A Caldwell; Jarrod A Call; Riccardo Calvani; Ana C Calvo; Miguel Calvo-Rubio Barrera; Niels Os Camara; Jacques H Camonis; Nadine Camougrand; Michelangelo Campanella; Edward M Campbell; François-Xavier Campbell-Valois; Silvia Campello; Ilaria Campesi; Juliane C Campos; Olivier Camuzard; Jorge Cancino; Danilo Candido de Almeida; Laura Canesi; Isabella Caniggia; Barbara Canonico; Carles Cantí; Bin Cao; Michele Caraglia; Beatriz Caramés; Evie H Carchman; Elena Cardenal-Muñoz; Cesar Cardenas; Luis Cardenas; Sandra M Cardoso; Jennifer S Carew; Georges F Carle; Gillian Carleton; Silvia Carloni; Didac Carmona-Gutierrez; Leticia A Carneiro; Oliana Carnevali; Julian M Carosi; Serena Carra; Alice Carrier; Lucie Carrier; Bernadette Carroll; A Brent Carter; Andreia Neves Carvalho; Magali Casanova; Caty Casas; Josefina Casas; Chiara Cassioli; Eliseo F Castillo; Karen Castillo; Sonia Castillo-Lluva; Francesca Castoldi; Marco Castori; Ariel F Castro; Margarida Castro-Caldas; Javier Castro-Hernandez; Susana Castro-Obregon; Sergio D Catz; Claudia Cavadas; Federica Cavaliere; Gabriella Cavallini; Maria Cavinato; Maria L Cayuela; Paula Cebollada Rica; Valentina Cecarini; Francesco Cecconi; Marzanna Cechowska-Pasko; Simone Cenci; Victòria Ceperuelo-Mallafré; João J Cerqueira; Janete M Cerutti; Davide Cervia; Vildan Bozok Cetintas; Silvia Cetrullo; Han-Jung Chae; Andrei S Chagin; Chee-Yin Chai; Gopal Chakrabarti; Oishee Chakrabarti; Tapas Chakraborty; Trinad Chakraborty; Mounia Chami; Georgios Chamilos; David W Chan; Edmond Y W Chan; Edward D Chan; H Y Edwin Chan; Helen H Chan; Hung Chan; Matthew T V Chan; Yau Sang Chan; Partha K Chandra; Chih-Peng Chang; Chunmei Chang; Hao-Chun Chang; Kai Chang; Jie Chao; Tracey Chapman; Nicolas Charlet-Berguerand; Samrat Chatterjee; Shail K Chaube; Anu Chaudhary; Santosh Chauhan; Edward Chaum; Frédéric Checler; Michael E Cheetham; Chang-Shi Chen; Guang-Chao Chen; Jian-Fu Chen; Liam L Chen; Leilei Chen; Lin Chen; Mingliang Chen; Mu-Kuan Chen; Ning Chen; Quan Chen; Ruey-Hwa Chen; Shi Chen; Wei Chen; Weiqiang Chen; Xin-Ming Chen; Xiong-Wen Chen; Xu Chen; Yan Chen; Ye-Guang Chen; Yingyu Chen; Yongqiang Chen; Yu-Jen Chen; Yue-Qin Chen; Zhefan Stephen Chen; Zhi Chen; Zhi-Hua Chen; Zhijian J Chen; Zhixiang Chen; Hanhua Cheng; Jun Cheng; Shi-Yuan Cheng; Wei Cheng; Xiaodong Cheng; Xiu-Tang Cheng; Yiyun Cheng; Zhiyong Cheng; Zhong Chen; Heesun Cheong; Jit Kong Cheong; Boris V Chernyak; Sara Cherry; Chi Fai Randy Cheung; Chun Hei Antonio Cheung; King-Ho Cheung; Eric Chevet; Richard J Chi; Alan Kwok Shing Chiang; Ferdinando Chiaradonna; Roberto Chiarelli; Mario Chiariello; Nathalia Chica; Susanna Chiocca; Mario Chiong; Shih-Hwa Chiou; Abhilash I Chiramel; Valerio Chiurchiù; Dong-Hyung Cho; Seong-Kyu Choe; Augustine M K Choi; Mary E Choi; Kamalika Roy Choudhury; Norman S Chow; Charleen T Chu; Jason P Chua; John Jia En Chua; Hyewon Chung; Kin Pan Chung; Seockhoon Chung; So-Hyang Chung; Yuen-Li Chung; Valentina Cianfanelli; Iwona A Ciechomska; Mariana Cifuentes; Laura Cinque; Sebahattin Cirak; Mara Cirone; Michael J Clague; Robert Clarke; Emilio Clementi; Eliana M Coccia; Patrice Codogno; Ehud Cohen; Mickael M Cohen; Tania Colasanti; Fiorella Colasuonno; Robert A Colbert; Anna Colell; Miodrag Čolić; Nuria S Coll; Mark O Collins; María I Colombo; Daniel A Colón-Ramos; Lydie Combaret; Sergio Comincini; Márcia R Cominetti; Antonella Consiglio; Andrea Conte; Fabrizio Conti; Viorica Raluca Contu; Mark R Cookson; Kevin M Coombs; Isabelle Coppens; Maria Tiziana Corasaniti; Dale P Corkery; Nils Cordes; Katia Cortese; Maria do Carmo Costa; Sarah Costantino; Paola Costelli; Ana Coto-Montes; Peter J Crack; Jose L Crespo; Alfredo Criollo; Valeria Crippa; Riccardo Cristofani; Tamas Csizmadia; Antonio Cuadrado; Bing Cui; Jun Cui; Yixian Cui; Yong Cui; Emmanuel Culetto; Andrea C Cumino; Andrey V Cybulsky; Mark J Czaja; Stanislaw J Czuczwar; Stefania D'Adamo; Marcello D'Amelio; Daniela D'Arcangelo; Andrew C D'Lugos; Gabriella D'Orazi; James A da Silva; Hormos Salimi Dafsari; Ruben K Dagda; Yasin Dagdas; Maria Daglia; Xiaoxia Dai; Yun Dai; Yuyuan Dai; Jessica Dal Col; Paul Dalhaimer; Luisa Dalla Valle; Tobias Dallenga; Guillaume Dalmasso; Markus Damme; Ilaria Dando; Nico P Dantuma; April L Darling; Hiranmoy Das; Srinivasan Dasarathy; Santosh K Dasari; Srikanta Dash; Oliver Daumke; Adrian N Dauphinee; Jeffrey S Davies; Valeria A Dávila; Roger J Davis; Tanja Davis; Sharadha Dayalan Naidu; Francesca De Amicis; Karolien De Bosscher; Francesca De Felice; Lucia De Franceschi; Chiara De Leonibus; Mayara G de Mattos Barbosa; Guido R Y De Meyer; Angelo De Milito; Cosimo De Nunzio; Clara De Palma; Mauro De Santi; Claudio De Virgilio; Daniela De Zio; Jayanta Debnath; Brian J DeBosch; Jean-Paul Decuypere; Mark A Deehan; Gianluca Deflorian; James DeGregori; Benjamin Dehay; Gabriel Del Rio; Joe R Delaney; Lea M D Delbridge; Elizabeth Delorme-Axford; M Victoria Delpino; Francesca Demarchi; Vilma Dembitz; Nicholas D Demers; Hongbin Deng; Zhiqiang Deng; Joern Dengjel; Paul Dent; Donna Denton; Melvin L DePamphilis; Channing J Der; Vojo Deretic; Albert Descoteaux; Laura Devis; Sushil Devkota; Olivier Devuyst; Grant Dewson; Mahendiran Dharmasivam; Rohan Dhiman; Diego di Bernardo; Manlio Di Cristina; Fabio Di Domenico; Pietro Di Fazio; Alessio Di Fonzo; Giovanni Di Guardo; Gianni M Di Guglielmo; Luca Di Leo; Chiara Di Malta; Alessia Di Nardo; Martina Di Rienzo; Federica Di Sano; George Diallinas; Jiajie Diao; Guillermo Diaz-Araya; Inés Díaz-Laviada; Jared M Dickinson; Marc Diederich; Mélanie Dieudé; Ivan Dikic; Shiping Ding; Wen-Xing Ding; Luciana Dini; Jelena Dinić; Miroslav Dinic; Albena T Dinkova-Kostova; Marc S Dionne; Jörg H W Distler; Abhinav Diwan; Ian M C Dixon; Mojgan Djavaheri-Mergny; Ina Dobrinski; Oxana Dobrovinskaya; Radek Dobrowolski; Renwick C J Dobson; Jelena Đokić; Serap Dokmeci Emre; Massimo Donadelli; Bo Dong; Xiaonan Dong; Zhiwu Dong; Gerald W Dorn Ii; Volker Dotsch; Huan Dou; Juan Dou; Moataz Dowaidar; Sami Dridi; Liat Drucker; Ailian Du; Caigan Du; Guangwei Du; Hai-Ning Du; Li-Lin Du; André du Toit; Shao-Bin Duan; Xiaoqiong Duan; Sónia P Duarte; Anna Dubrovska; Elaine A Dunlop; Nicolas Dupont; Raúl V Durán; Bilikere S Dwarakanath; Sergey A Dyshlovoy; Darius Ebrahimi-Fakhari; Leopold Eckhart; Charles L Edelstein; Thomas Efferth; Eftekhar Eftekharpour; Ludwig Eichinger; Nabil Eid; Tobias Eisenberg; N Tony Eissa; Sanaa Eissa; Miriam Ejarque; Abdeljabar El Andaloussi; Nazira El-Hage; Shahenda El-Naggar; Anna Maria Eleuteri; Eman S El-Shafey; Mohamed Elgendy; Aristides G Eliopoulos; María M Elizalde; Philip M Elks; Hans-Peter Elsasser; Eslam S Elsherbiny; Brooke M Emerling; N C Tolga Emre; Christina H Eng; Nikolai Engedal; Anna-Mart Engelbrecht; Agnete S T Engelsen; Jorrit M Enserink; Ricardo Escalante; Audrey Esclatine; Mafalda Escobar-Henriques; Eeva-Liisa Eskelinen; Lucile Espert; Makandjou-Ola Eusebio; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Francesco Facchiano; Bengt Fadeel; Claudio Fader; Alex C Faesen; W Douglas Fairlie; Alberto Falcó; Bjorn H Falkenburger; Daping Fan; Jie Fan; Yanbo Fan; Evandro F Fang; Yanshan Fang; Yognqi Fang; Manolis Fanto; Tamar Farfel-Becker; Mathias Faure; Gholamreza Fazeli; Anthony O Fedele; Arthur M Feldman; Du Feng; Jiachun Feng; Lifeng Feng; Yibin Feng; Yuchen Feng; Wei Feng; Thais Fenz Araujo; Thomas A Ferguson; Álvaro F Fernández; Jose C Fernandez-Checa; Sonia Fernández-Veledo; Alisdair R Fernie; Anthony W Ferrante; Alessandra Ferraresi; Merari F Ferrari; Julio C B Ferreira; Susan Ferro-Novick; Antonio Figueras; Riccardo Filadi; Nicoletta Filigheddu; Eduardo Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; Vittorio Fineschi; Francesca Finetti; Steven Finkbeiner; Edward A Fisher; Paul B Fisher; Flavio Flamigni; Steven J Fliesler; Trude H Flo; Ida Florance; Oliver Florey; Tullio Florio; Erika Fodor; Carlo Follo; Edward A Fon; Antonella Forlino; Francesco Fornai; Paola Fortini; Anna Fracassi; Alessandro Fraldi; Brunella Franco; Rodrigo Franco; Flavia Franconi; Lisa B Frankel; Scott L Friedman; Leopold F Fröhlich; Gema Frühbeck; Jose M Fuentes; Yukio Fujiki; Naonobu Fujita; Yuuki Fujiwara; Mitsunori Fukuda; Simone Fulda; Luc Furic; Norihiko Furuya; Carmela Fusco; Michaela U Gack; Lidia Gaffke; Sehamuddin Galadari; Alessia Galasso; Maria F Galindo; Sachith Gallolu Kankanamalage; Lorenzo Galluzzi; Vincent Galy; Noor Gammoh; Boyi Gan; Ian G Ganley; Feng Gao; Hui Gao; Minghui Gao; Ping Gao; Shou-Jiang Gao; Wentao Gao; Xiaobo Gao; Ana Garcera; Maria Noé Garcia; Verónica E Garcia; Francisco García-Del Portillo; Vega Garcia-Escudero; Aracely Garcia-Garcia; Marina Garcia-Macia; Diana García-Moreno; Carmen Garcia-Ruiz; Patricia García-Sanz; Abhishek D Garg; Ricardo Gargini; Tina Garofalo; Robert F Garry; Nils C Gassen; Damian Gatica; Liang Ge; Wanzhong Ge; Ruth Geiss-Friedlander; Cecilia Gelfi; Pascal Genschik; Ian E Gentle; Valeria Gerbino; Christoph Gerhardt; Kyla Germain; Marc Germain; David A Gewirtz; Elham Ghasemipour Afshar; Saeid Ghavami; Alessandra Ghigo; Manosij Ghosh; Georgios Giamas; Claudia Giampietri; Alexandra Giatromanolaki; Gary E Gibson; Spencer B Gibson; Vanessa Ginet; Edward Giniger; Carlotta Giorgi; Henrique Girao; Stephen E Girardin; Mridhula Giridharan; Sandy Giuliano; Cecilia Giulivi; Sylvie Giuriato; Julien Giustiniani; Alexander Gluschko; Veit Goder; Alexander Goginashvili; Jakub Golab; David C Goldstone; Anna Golebiewska; Luciana R Gomes; Rodrigo Gomez; Rubén Gómez-Sánchez; Maria Catalina Gomez-Puerto; Raquel Gomez-Sintes; Qingqiu Gong; Felix M Goni; Javier González-Gallego; Tomas Gonzalez-Hernandez; Rosa A Gonzalez-Polo; Jose A Gonzalez-Reyes; Patricia González-Rodríguez; Ing Swie Goping; Marina S Gorbatyuk; Nikolai V Gorbunov; Kıvanç Görgülü; Roxana M Gorojod; Sharon M Gorski; Sandro Goruppi; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Martin Graef; Markus H Gräler; Veronica Granatiero; Daniel Grasso; Joshua P Gray; Douglas R Green; Alexander Greenhough; Stephen L Gregory; Edward F Griffin; Mark W Grinstaff; Frederic Gros; Charles Grose; Angelina S Gross; Florian Gruber; Paolo Grumati; Tilman Grune; Xueyan Gu; Jun-Lin Guan; Carlos M Guardia; Kishore Guda; Flora Guerra; Consuelo Guerri; Prasun Guha; Carlos Guillén; Shashi Gujar; Anna Gukovskaya; Ilya Gukovsky; Jan Gunst; Andreas Günther; Anyonya R Guntur; Chuanyong Guo; Chun Guo; Hongqing Guo; Lian-Wang Guo; Ming Guo; Pawan Gupta; Shashi Kumar Gupta; Swapnil Gupta; Veer Bala Gupta; Vivek Gupta; Asa B Gustafsson; David D Gutterman; Ranjitha H B; Annakaisa Haapasalo; James E Haber; Aleksandra Hać; Shinji Hadano; Anders J Hafrén; Mansour Haidar; Belinda S Hall; Gunnel Halldén; Anne Hamacher-Brady; Andrea Hamann; Maho Hamasaki; Weidong Han; Malene Hansen; Phyllis I Hanson; Zijian Hao; Masaru Harada; Ljubica Harhaji-Trajkovic; Nirmala Hariharan; Nigil Haroon; James Harris; Takafumi Hasegawa; Noor Hasima Nagoor; Jeffrey A Haspel; Volker Haucke; Wayne D Hawkins; Bruce A Hay; Cole M Haynes; Soren B Hayrabedyan; Thomas S Hays; Congcong He; Qin He; Rong-Rong He; You-Wen He; Yu-Ying He; Yasser Heakal; Alexander M Heberle; J Fielding Hejtmancik; Gudmundur Vignir Helgason; Vanessa Henkel; Marc Herb; Alexander Hergovich; Anna Herman-Antosiewicz; Agustín Hernández; Carlos Hernandez; Sergio Hernandez-Diaz; Virginia Hernandez-Gea; Amaury Herpin; Judit Herreros; Javier H Hervás; Daniel Hesselson; Claudio Hetz; Volker T Heussler; Yujiro Higuchi; Sabine Hilfiker; Joseph A Hill; William S Hlavacek; Emmanuel A Ho; Idy H T Ho; Philip Wing-Lok Ho; Shu-Leong Ho; Wan Yun Ho; G Aaron Hobbs; Mark Hochstrasser; Peter H M Hoet; Daniel Hofius; Paul Hofman; Annika Höhn; Carina I Holmberg; Jose R Hombrebueno; Chang-Won Hong Yi-Ren Hong; Lora V Hooper; Thorsten Hoppe; Rastislav Horos; Yujin Hoshida; I-Lun Hsin; Hsin-Yun Hsu; Bing Hu; Dong Hu; Li-Fang Hu; Ming Chang Hu; Ronggui Hu; Wei Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Jinlian Hua; Yingqi Hua; Chongmin Huan; Canhua Huang; Chuanshu Huang; Chuanxin Huang; Chunling Huang; Haishan Huang; Kun Huang; Michael L H Huang; Rui Huang; Shan Huang; Tianzhi Huang; Xing Huang; Yuxiang Jack Huang; Tobias B Huber; Virginie Hubert; Christian A Hubner; Stephanie M Hughes; William E Hughes; Magali Humbert; Gerhard Hummer; James H Hurley; Sabah Hussain; Salik Hussain; Patrick J Hussey; Martina Hutabarat; Hui-Yun Hwang; Seungmin Hwang; Antonio Ieni; Fumiyo Ikeda; Yusuke Imagawa; Yuzuru Imai; Carol Imbriano; Masaya Imoto; Denise M Inman; Ken Inoki; Juan Iovanna; Renato V Iozzo; Giuseppe Ippolito; Javier E Irazoqui; Pablo Iribarren; Mohd Ishaq; Makoto Ishikawa; Nestor Ishimwe; Ciro Isidoro; Nahed Ismail; Shohreh Issazadeh-Navikas; Eisuke Itakura; Daisuke Ito; Davor Ivankovic; Saška Ivanova; Anand Krishnan V Iyer; José M Izquierdo; Masanori Izumi; Marja Jäättelä; Majid Sakhi Jabir; William T Jackson; Nadia Jacobo-Herrera; Anne-Claire Jacomin; Elise Jacquin; Pooja Jadiya; Hartmut Jaeschke; Chinnaswamy Jagannath; Arjen J Jakobi; Johan Jakobsson; Bassam Janji; Pidder Jansen-Dürr; Patric J Jansson; Jonathan Jantsch; Sławomir Januszewski; Alagie Jassey; Steve Jean; Hélène Jeltsch-David; Pavla Jendelova; Andreas Jenny; Thomas E Jensen; Niels Jessen; Jenna L Jewell; Jing Ji; Lijun Jia; Rui Jia; Liwen Jiang; Qing Jiang; Richeng Jiang; Teng Jiang; Xuejun Jiang; Yu Jiang; Maria Jimenez-Sanchez; Eun-Jung Jin; Fengyan Jin; Hongchuan Jin; Li Jin; Luqi Jin; Meiyan Jin; Si Jin; Eun-Kyeong Jo; Carine Joffre; Terje Johansen; Gail V W Johnson; Simon A Johnston; Eija Jokitalo; Mohit Kumar Jolly; Leo A B Joosten; Joaquin Jordan; Bertrand Joseph; Dianwen Ju; Jeong-Sun Ju; Jingfang Ju; Esmeralda Juárez; Delphine Judith; Gábor Juhász; Youngsoo Jun; Chang Hwa Jung; Sung-Chul Jung; Yong Keun Jung; Heinz Jungbluth; Johannes Jungverdorben; Steffen Just; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Daniel Kaganovich; Alon Kahana; Renate Kain; Shinjo Kajimura; Maria Kalamvoki; Manjula Kalia; Danuta S Kalinowski; Nina Kaludercic; Ioanna Kalvari; Joanna Kaminska; Vitaliy O Kaminskyy; Hiromitsu Kanamori; Keizo Kanasaki; Chanhee Kang; Rui Kang; Sang Sun Kang; Senthilvelrajan Kaniyappan; Tomotake Kanki; Thirumala-Devi Kanneganti; Anumantha G Kanthasamy; Arthi Kanthasamy; Marc Kantorow; Orsolya Kapuy; Michalis V Karamouzis; Md Razaul Karim; Parimal Karmakar; Rajesh G Katare; Masaru Kato; Stefan H E Kaufmann; Anu Kauppinen; Gur P Kaushal; Susmita Kaushik; Kiyoshi Kawasaki; Kemal Kazan; Po-Yuan Ke; Damien J Keating; Ursula Keber; John H Kehrl; Kate E Keller; Christian W Keller; Jongsook Kim Kemper; Candia M Kenific; Oliver Kepp; Stephanie Kermorgant; Andreas Kern; Robin Ketteler; Tom G Keulers; Boris Khalfin; Hany Khalil; Bilon Khambu; Shahid Y Khan; Vinoth Kumar Megraj Khandelwal; Rekha Khandia; Widuri Kho; Noopur V Khobrekar; Sataree Khuansuwan; Mukhran Khundadze; Samuel A Killackey; Dasol Kim; Deok Ryong Kim; Do-Hyung Kim; Dong-Eun Kim; Eun Young Kim; Eun-Kyoung Kim; Hak-Rim Kim; Hee-Sik Kim; Jeong Hun Kim; Jin Kyung Kim; Jin-Hoi Kim; Joungmok Kim; Ju Hwan Kim; Keun Il Kim; Peter K Kim; Seong-Jun Kim; Scot R Kimball; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Matthew A King; Kerri J Kinghorn; Conan G Kinsey; Vladimir Kirkin; Lorrie A Kirshenbaum; Sergey L Kiselev; Shuji Kishi; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Richard N Kitsis; Josef T Kittler; Ole Kjaerulff; Peter S Klein; Thomas Klopstock; Jochen Klucken; Helene Knævelsrud; Roland L Knorr; Ben C B Ko; Fred Ko; Jiunn-Liang Ko; Hotaka Kobayashi; Satoru Kobayashi; Ina Koch; Jan C Koch; Ulrich Koenig; Donat Kögel; Young Ho Koh; Masato Koike; Sepp D Kohlwein; Nur M Kocaturk; Masaaki Komatsu; Jeannette König; Toru Kono; Benjamin T Kopp; Tamas Korcsmaros; Gözde Korkmaz; Viktor I Korolchuk; Mónica Suárez Korsnes; Ali Koskela; Janaiah Kota; Yaichiro Kotake; Monica L Kotler; Yanjun Kou; Michael I Koukourakis; Evangelos Koustas; Attila L Kovacs; Tibor Kovács; Daisuke Koya; Tomohiro Kozako; Claudine Kraft; Dimitri Krainc; Helmut Krämer; Anna D Krasnodembskaya; Carole Kretz-Remy; Guido Kroemer; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Sabine Kuenen; Lars Kuerschner; Thomas Kukar; Ajay Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Sharad Kumar; Shinji Kume; Caroline Kumsta; Chanakya N Kundu; Mondira Kundu; Ajaikumar B Kunnumakkara; Lukasz Kurgan; Tatiana G Kutateladze; Ozlem Kutlu; SeongAe Kwak; Ho Jeong Kwon; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert La Spada; Patrick Labonté; Sylvain Ladoire; Ilaria Laface; Frank Lafont; Diane C Lagace; Vikramjit Lahiri; Zhibing Lai; Angela S Laird; Aparna Lakkaraju; Trond Lamark; Sheng-Hui Lan; Ane Landajuela; Darius J R Lane; Jon D Lane; Charles H Lang; Carsten Lange; Ülo Langel; Rupert Langer; Pierre Lapaquette; Jocelyn Laporte; Nicholas F LaRusso; Isabel Lastres-Becker; Wilson Chun Yu Lau; Gordon W Laurie; Sergio Lavandero; Betty Yuen Kwan Law; Helen Ka-Wai Law; Rob Layfield; Weidong Le; Herve Le Stunff; Alexandre Y Leary; Jean-Jacques Lebrun; Lionel Y W Leck; Jean-Philippe Leduc-Gaudet; Changwook Lee; Chung-Pei Lee; Da-Hye Lee; Edward B Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Heung Kyu Lee; Jae Man Lee; Jason S Lee; Jin-A Lee; Joo-Yong Lee; Jun Hee Lee; Michael Lee; Min Goo Lee; Min Jae Lee; Myung-Shik Lee; Sang Yoon Lee; Seung-Jae Lee; Stella Y Lee; Sung Bae Lee; Won Hee Lee; Ying-Ray Lee; Yong-Ho Lee; Youngil Lee; Christophe Lefebvre; Renaud Legouis; Yu L Lei; Yuchen Lei; Sergey Leikin; Gerd Leitinger; Leticia Lemus; Shuilong Leng; Olivia Lenoir; Guido Lenz; Heinz Josef Lenz; Paola Lenzi; Yolanda León; Andréia M Leopoldino; Christoph Leschczyk; Stina Leskelä; Elisabeth Letellier; Chi-Ting Leung; Po Sing Leung; Jeremy S Leventhal; Beth Levine; Patrick A Lewis; Klaus Ley; Bin Li; Da-Qiang Li; Jianming Li; Jing Li; Jiong Li; Ke Li; Liwu Li; Mei Li; Min Li; Min Li; Ming Li; Mingchuan Li; Pin-Lan Li; Ming-Qing Li; Qing Li; Sheng Li; Tiangang Li; Wei Li; Wenming Li; Xue Li; Yi-Ping Li; Yuan Li; Zhiqiang Li; Zhiyong Li; Zhiyuan Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Weicheng Liang; Yongheng Liang; YongTian Liang; Guanghong Liao; Lujian Liao; Mingzhi Liao; Yung-Feng Liao; Mariangela Librizzi; Pearl P Y Lie; Mary A Lilly; Hyunjung J Lim; Thania R R Lima; Federica Limana; Chao Lin; Chih-Wen Lin; Dar-Shong Lin; Fu-Cheng Lin; Jiandie D Lin; Kurt M Lin; Kwang-Huei Lin; Liang-Tzung Lin; Pei-Hui Lin; Qiong Lin; Shaofeng Lin; Su-Ju Lin; Wenyu Lin; Xueying Lin; Yao-Xin Lin; Yee-Shin Lin; Rafael Linden; Paula Lindner; Shuo-Chien Ling; Paul Lingor; Amelia K Linnemann; Yih-Cherng Liou; Marta M Lipinski; Saška Lipovšek; Vitor A Lira; Natalia Lisiak; Paloma B Liton; Chao Liu; Ching-Hsuan Liu; Chun-Feng Liu; Cui Hua Liu; Fang Liu; Hao Liu; Hsiao-Sheng Liu; Hua-Feng Liu; Huifang Liu; Jia Liu; Jing Liu; Julia Liu; Leyuan Liu; Longhua Liu; Meilian Liu; Qin Liu; Wei Liu; Wende Liu; Xiao-Hong Liu; Xiaodong Liu; Xingguo Liu; Xu Liu; Xuedong Liu; Yanfen Liu; Yang Liu; Yang Liu; Yueyang Liu; Yule Liu; J Andrew Livingston; Gerard Lizard; Jose M Lizcano; Senka Ljubojevic-Holzer; Matilde E LLeonart; David Llobet-Navàs; Alicia Llorente; Chih Hung Lo; Damián Lobato-Márquez; Qi Long; Yun Chau Long; Ben Loos; Julia A Loos; Manuela G López; Guillermo López-Doménech; José Antonio López-Guerrero; Ana T López-Jiménez; Óscar López-Pérez; Israel López-Valero; Magdalena J Lorenowicz; Mar Lorente; Peter Lorincz; Laura Lossi; Sophie Lotersztajn; Penny E Lovat; Jonathan F Lovell; Alenka Lovy; Péter Lőw; Guang Lu; Haocheng Lu; Jia-Hong Lu; Jin-Jian Lu; Mengji Lu; Shuyan Lu; Alessandro Luciani; John M Lucocq; Paula Ludovico; Micah A Luftig; Morten Luhr; Diego Luis-Ravelo; Julian J Lum; Liany Luna-Dulcey; Anders H Lund; Viktor K Lund; Jan D Lünemann; Patrick Lüningschrör; Honglin Luo; Rongcan Luo; Shouqing Luo; Zhi Luo; Claudio Luparello; Bernhard Lüscher; Luan Luu; Alex Lyakhovich; Konstantin G Lyamzaev; Alf Håkon Lystad; Lyubomyr Lytvynchuk; Alvin C Ma; Changle Ma; Mengxiao Ma; Ning-Fang Ma; Quan-Hong Ma; Xinliang Ma; Yueyun Ma; Zhenyi Ma; Ormond A MacDougald; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; Sandra Maday; Frank Madeo; Muniswamy Madesh; Tobias Madl; Julio Madrigal-Matute; Akiko Maeda; Yasuhiro Maejima; Marta Magarinos; Poornima Mahavadi; Emiliano Maiani; Kenneth Maiese; Panchanan Maiti; Maria Chiara Maiuri; Barbara Majello; Michael B Major; Elena Makareeva; Fayaz Malik; Karthik Mallilankaraman; Walter Malorni; Alina Maloyan; Najiba Mammadova; Gene Chi Wai Man; Federico Manai; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Masoud H Manjili; Ravi Manjithaya; Patricio Manque; Bella B Manshian; Raquel Manzano; Claudia Manzoni; Kai Mao; Cinzia Marchese; Sandrine Marchetti; Anna Maria Marconi; Fabrizio Marcucci; Stefania Mardente; Olga A Mareninova; Marta Margeta; Muriel Mari; Sara Marinelli; Oliviero Marinelli; Guillermo Mariño; Sofia Mariotto; Richard S Marshall; Mark R Marten; Sascha Martens; Alexandre P J Martin; Katie R Martin; Sara Martin; Shaun Martin; Adrián Martín-Segura; Miguel A Martín-Acebes; Inmaculada Martin-Burriel; Marcos Martin-Rincon; Paloma Martin-Sanz; José A Martina; Wim Martinet; Aitor Martinez; Ana Martinez; Jennifer Martinez; Moises Martinez Velazquez; Nuria Martinez-Lopez; Marta Martinez-Vicente; Daniel O Martins; Joilson O Martins; Waleska K Martins; Tania Martins-Marques; Emanuele Marzetti; Shashank Masaldan; Celine Masclaux-Daubresse; Douglas G Mashek; Valentina Massa; Lourdes Massieu; Glenn R Masson; Laura Masuelli; Anatoliy I Masyuk; Tetyana V Masyuk; Paola Matarrese; Ander Matheu; Satoaki Matoba; Sachiko Matsuzaki; Pamela Mattar; Alessandro Matte; Domenico Mattoscio; José L Mauriz; Mario Mauthe; Caroline Mauvezin; Emanual Maverakis; Paola Maycotte; Johanna Mayer; Gianluigi Mazzoccoli; Cristina Mazzoni; Joseph R Mazzulli; Nami McCarty; Christine McDonald; Mitchell R McGill; Sharon L McKenna; BethAnn McLaughlin; Fionn McLoughlin; Mark A McNiven; Thomas G McWilliams; Fatima Mechta-Grigoriou; Tania Catarina Medeiros; Diego L Medina; Lynn A Megeney; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Alfred J Meijer; Annemarie H Meijer; Jakob Mejlvang; Alicia Meléndez; Annette Melk; Gonen Memisoglu; Alexandrina F Mendes; Delong Meng; Fei Meng; Tian Meng; Rubem Menna-Barreto; Manoj B Menon; Carol Mercer; Anne E Mercier; Jean-Louis Mergny; Adalberto Merighi; Seth D Merkley; Giuseppe Merla; Volker Meske; Ana Cecilia Mestre; Shree Padma Metur; Christian Meyer; Hemmo Meyer; Wenyi Mi; Jeanne Mialet-Perez; Junying Miao; Lucia Micale; Yasuo Miki; Enrico Milan; Małgorzata Milczarek; Dana L Miller; Samuel I Miller; Silke Miller; Steven W Millward; Ira Milosevic; Elena A Minina; Hamed Mirzaei; Hamid Reza Mirzaei; Mehdi Mirzaei; Amit Mishra; Nandita Mishra; Paras Kumar Mishra; Maja Misirkic Marjanovic; Roberta Misasi; Amit Misra; Gabriella Misso; Claire Mitchell; Geraldine Mitou; Tetsuji Miura; Shigeki Miyamoto; Makoto Miyazaki; Mitsunori Miyazaki; Taiga Miyazaki; Keisuke Miyazawa; Noboru Mizushima; Trine H Mogensen; Baharia Mograbi; Reza Mohammadinejad; Yasir Mohamud; Abhishek Mohanty; Sipra Mohapatra; Torsten Möhlmann; Asif Mohmmed; Anna Moles; Kelle H Moley; Maurizio Molinari; Vincenzo Mollace; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Costanza Montagna; Mervyn J Monteiro; Andrea Montella; L Ruth Montes; Barbara Montico; Vinod K Mony; Giacomo Monzio Compagnoni; Michael N Moore; Mohammad A Moosavi; Ana L Mora; Marina Mora; David Morales-Alamo; Rosario Moratalla; Paula I Moreira; Elena Morelli; Sandra Moreno; Daniel Moreno-Blas; Viviana Moresi; Benjamin Morga; Alwena H Morgan; Fabrice Morin; Hideaki Morishita; Orson L Moritz; Mariko Moriyama; Yuji Moriyasu; Manuela Morleo; Eugenia Morselli; Jose F Moruno-Manchon; Jorge Moscat; Serge Mostowy; Elisa Motori; Andrea Felinto Moura; Naima Moustaid-Moussa; Maria Mrakovcic; Gabriel Muciño-Hernández; Anupam Mukherjee; Subhadip Mukhopadhyay; Jean M Mulcahy Levy; Victoriano Mulero; Sylviane Muller; Christian Münch; Ashok Munjal; Pura Munoz-Canoves; Teresa Muñoz-Galdeano; Christian Münz; Tomokazu Murakawa; Claudia Muratori; Brona M Murphy; J Patrick Murphy; Aditya Murthy; Timo T Myöhänen; Indira U Mysorekar; Jennifer Mytych; Seyed Mohammad Nabavi; Massimo Nabissi; Péter Nagy; Jihoon Nah; Aimable Nahimana; Ichiro Nakagawa; Ken Nakamura; Hitoshi Nakatogawa; Shyam S Nandi; Meera Nanjundan; Monica Nanni; Gennaro Napolitano; Roberta Nardacci; Masashi Narita; Melissa Nassif; Ilana Nathan; Manabu Natsumeda; Ryno J Naude; Christin Naumann; Olaia Naveiras; Fatemeh Navid; Steffan T Nawrocki; Taras Y Nazarko; Francesca Nazio; Florentina Negoita; Thomas Neill; Amanda L Neisch; Luca M Neri; Mihai G Netea; Patrick Neubert; Thomas P Neufeld; Dietbert Neumann; Albert Neutzner; Phillip T Newton; Paul A Ney; Ioannis P Nezis; Charlene C W Ng; Tzi Bun Ng; Hang T T Nguyen; Long T Nguyen; Hong-Min Ni; Clíona Ní Cheallaigh; Zhenhong Ni; M Celeste Nicolao; Francesco Nicoli; Manuel Nieto-Diaz; Per Nilsson; Shunbin Ning; Rituraj Niranjan; Hiroshi Nishimune; Mireia Niso-Santano; Ralph A Nixon; Annalisa Nobili; Clevio Nobrega; Takeshi Noda; Uxía Nogueira-Recalde; Trevor M Nolan; Ivan Nombela; Ivana Novak; Beatriz Novoa; Takashi Nozawa; Nobuyuki Nukina; Carmen Nussbaum-Krammer; Jesper Nylandsted; Tracey R O'Donovan; Seónadh M O'Leary; Eyleen J O'Rourke; Mary P O'Sullivan; Timothy E O'Sullivan; Salvatore Oddo; Ina Oehme; Michinaga Ogawa; Eric Ogier-Denis; Margret H Ogmundsdottir; Besim Ogretmen; Goo Taeg Oh; Seon-Hee Oh; Young J Oh; Takashi Ohama; Yohei Ohashi; Masaki Ohmuraya; Vasileios Oikonomou; Rani Ojha; Koji Okamoto; Hitoshi Okazawa; Masahide Oku; Sara Oliván; Jorge M A Oliveira; Michael Ollmann; James A Olzmann; Shakib Omari; M Bishr Omary; Gizem Önal; Martin Ondrej; Sang-Bing Ong; Sang-Ging Ong; Anna Onnis; Juan A Orellana; Sara Orellana-Muñoz; Maria Del Mar Ortega-Villaizan; Xilma R Ortiz-Gonzalez; Elena Ortona; Heinz D Osiewacz; Abdel-Hamid K Osman; Rosario Osta; Marisa S Otegui; Kinya Otsu; Christiane Ott; Luisa Ottobrini; Jing-Hsiung James Ou; Tiago F Outeiro; Inger Oynebraten; Melek Ozturk; Gilles Pagès; Susanta Pahari; Marta Pajares; Utpal B Pajvani; Rituraj Pal; Simona Paladino; Nicolas Pallet; Michela Palmieri; Giuseppe Palmisano; Camilla Palumbo; Francesco Pampaloni; Lifeng Pan; Qingjun Pan; Wenliang Pan; Xin Pan; Ganna Panasyuk; Rahul Pandey; Udai B Pandey; Vrajesh Pandya; Francesco Paneni; Shirley Y Pang; Elisa Panzarini; Daniela L Papademetrio; Elena Papaleo; Daniel Papinski; Diana Papp; Eun Chan Park; Hwan Tae Park; Ji-Man Park; Jong-In Park; Joon Tae Park; Junsoo Park; Sang Chul Park; Sang-Youel Park; Abraham H Parola; Jan B Parys; Adrien Pasquier; Benoit Pasquier; João F Passos; Nunzia Pastore; Hemal H Patel; Daniel Patschan; Sophie Pattingre; Gustavo Pedraza-Alva; Jose Pedraza-Chaverri; Zully Pedrozo; Gang Pei; Jianming Pei; Hadas Peled-Zehavi; Joaquín M Pellegrini; Joffrey Pelletier; Miguel A Peñalva; Di Peng; Ying Peng; Fabio Penna; Maria Pennuto; Francesca Pentimalli; Cláudia Mf Pereira; Gustavo J S Pereira; Lilian C Pereira; Luis Pereira de Almeida; Nirma D Perera; Ángel Pérez-Lara; Ana B Perez-Oliva; María Esther Pérez-Pérez; Palsamy Periyasamy; Andras Perl; Cristiana Perrotta; Ida Perrotta; Richard G Pestell; Morten Petersen; Irina Petrache; Goran Petrovski; Thorsten Pfirrmann; Astrid S Pfister; Jennifer A Philips; Huifeng Pi; Anna Picca; Alicia M Pickrell; Sandy Picot; Giovanna M Pierantoni; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Karolina Pierzynowska; Federico Pietrocola; Miroslawa Pietruczuk; Claudio Pignata; Felipe X Pimentel-Muiños; Mario Pinar; Roberta O Pinheiro; Ronit Pinkas-Kramarski; Paolo Pinton; Karolina Pircs; Sujan Piya; Paola Pizzo; Theo S Plantinga; Harald W Platta; Ainhoa Plaza-Zabala; Markus Plomann; Egor Y Plotnikov; Helene Plun-Favreau; Ryszard Pluta; Roger Pocock; Stefanie Pöggeler; Christian Pohl; Marc Poirot; Angelo Poletti; Marisa Ponpuak; Hana Popelka; Blagovesta Popova; Helena Porta; Soledad Porte Alcon; Eliana Portilla-Fernandez; Martin Post; Malia B Potts; Joanna Poulton; Ted Powers; Veena Prahlad; Tomasz K Prajsnar; Domenico Praticò; Rosaria Prencipe; Muriel Priault; Tassula Proikas-Cezanne; Vasilis J Promponas; Christopher G Proud; Rosa Puertollano; Luigi Puglielli; Thomas Pulinilkunnil; Deepika Puri; Rajat Puri; Julien Puyal; Xiaopeng Qi; Yongmei Qi; Wenbin Qian; Lei Qiang; Yu Qiu; Joe Quadrilatero; Jorge Quarleri; Nina Raben; Hannah Rabinowich; Debora Ragona; Michael J Ragusa; Nader Rahimi; Marveh Rahmati; Valeria Raia; Nuno Raimundo; Namakkal-Soorappan Rajasekaran; Sriganesh Ramachandra Rao; Abdelhaq Rami; Ignacio Ramírez-Pardo; David B Ramsden; Felix Randow; Pundi N Rangarajan; Danilo Ranieri; Hai Rao; Lang Rao; Rekha Rao; Sumit Rathore; J Arjuna Ratnayaka; Edward A Ratovitski; Palaniyandi Ravanan; Gloria Ravegnini; Swapan K Ray; Babak Razani; Vito Rebecca; Fulvio Reggiori; Anne Régnier-Vigouroux; Andreas S Reichert; David Reigada; Jan H Reiling; Theo Rein; Siegfried Reipert; Rokeya Sultana Rekha; Hongmei Ren; Jun Ren; Weichao Ren; Tristan Renault; Giorgia Renga; Karen Reue; Kim Rewitz; Bruna Ribeiro de Andrade Ramos; S Amer Riazuddin; Teresa M Ribeiro-Rodrigues; Jean-Ehrland Ricci; Romeo Ricci; Victoria Riccio; Des R Richardson; Yasuko Rikihisa; Makarand V Risbud; Ruth M Risueño; Konstantinos Ritis; Salvatore Rizza; Rosario Rizzuto; Helen C Roberts; Luke D Roberts; Katherine J Robinson; Maria Carmela Roccheri; Stephane Rocchi; George G Rodney; Tiago Rodrigues; Vagner Ramon Rodrigues Silva; Amaia Rodriguez; Ruth Rodriguez-Barrueco; Nieves Rodriguez-Henche; Humberto Rodriguez-Rocha; Jeroen Roelofs; Robert S Rogers; Vladimir V Rogov; Ana I Rojo; Krzysztof Rolka; Vanina Romanello; Luigina Romani; Alessandra Romano; Patricia S Romano; David Romeo-Guitart; Luis C Romero; Montserrat Romero; Joseph C Roney; Christopher Rongo; Sante Roperto; Mathias T Rosenfeldt; Philip Rosenstiel; Anne G Rosenwald; Kevin A Roth; Lynn Roth; Steven Roth; Kasper M A Rouschop; Benoit D Roussel; Sophie Roux; Patrizia Rovere-Querini; Ajit Roy; Aurore Rozieres; Diego Ruano; David C Rubinsztein; Maria P Rubtsova; Klaus Ruckdeschel; Christoph Ruckenstuhl; Emil Rudolf; Rüdiger Rudolf; Alessandra Ruggieri; Avnika Ashok Ruparelia; Paola Rusmini; Ryan R Russell; Gian Luigi Russo; Maria Russo; Rossella Russo; Oxana O Ryabaya; Kevin M Ryan; Kwon-Yul Ryu; Maria Sabater-Arcis; Ulka Sachdev; Michael Sacher; Carsten Sachse; Abhishek Sadhu; Junichi Sadoshima; Nathaniel Safren; Paul Saftig; Antonia P Sagona; Gaurav Sahay; Amirhossein Sahebkar; Mustafa Sahin; Ozgur Sahin; Sumit Sahni; Nayuta Saito; Shigeru Saito; Tsunenori Saito; Ryohei Sakai; Yasuyoshi Sakai; Jun-Ichi Sakamaki; Kalle Saksela; Gloria Salazar; Anna Salazar-Degracia; Ghasem H Salekdeh; Ashok K Saluja; Belém Sampaio-Marques; Maria Cecilia Sanchez; Jose A Sanchez-Alcazar; Victoria Sanchez-Vera; Vanessa Sancho-Shimizu; J Thomas Sanderson; Marco Sandri; Stefano Santaguida; Laura Santambrogio; Magda M Santana; Giorgio Santoni; Alberto Sanz; Pascual Sanz; Shweta Saran; Marco Sardiello; Timothy J Sargeant; Apurva Sarin; Chinmoy Sarkar; Sovan Sarkar; Maria-Rosa Sarrias; Surajit Sarkar; Dipanka Tanu Sarmah; Jaakko Sarparanta; Aishwarya Sathyanarayan; Ranganayaki Sathyanarayanan; K Matthew Scaglione; Francesca Scatozza; Liliana Schaefer; Zachary T Schafer; Ulrich E Schaible; Anthony H V Schapira; Michael Scharl; Hermann M Schatzl; Catherine H Schein; Wiep Scheper; David Scheuring; Maria Vittoria Schiaffino; Monica Schiappacassi; Rainer Schindl; Uwe Schlattner; Oliver Schmidt; Roland Schmitt; Stephen D Schmidt; Ingo Schmitz; Eran Schmukler; Anja Schneider; Bianca E Schneider; Romana Schober; Alejandra C Schoijet; Micah B Schott; Michael Schramm; Bernd Schröder; Kai Schuh; Christoph Schüller; Ryan J Schulze; Lea Schürmanns; Jens C Schwamborn; Melanie Schwarten; Filippo Scialo; Sebastiano Sciarretta; Melanie J Scott; Kathleen W Scotto; A Ivana Scovassi; Andrea Scrima; Aurora Scrivo; David Sebastian; Salwa Sebti; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Iban Seiliez; Ekihiro Seki; Scott B Selleck; Frank W Sellke; Joshua T Selsby; Michael Sendtner; Serif Senturk; Elena Seranova; Consolato Sergi; Ruth Serra-Moreno; Hiromi Sesaki; Carmine Settembre; Subba Rao Gangi Setty; Gianluca Sgarbi; Ou Sha; John J Shacka; Javeed A Shah; Dantong Shang; Changshun Shao; Feng Shao; Soroush Sharbati; Lisa M Sharkey; Dipali Sharma; Gaurav Sharma; Kulbhushan Sharma; Pawan Sharma; Surendra Sharma; Han-Ming Shen; Hongtao Shen; Jiangang Shen; Ming Shen; Weili Shen; Zheni Shen; Rui Sheng; Zhi Sheng; Zu-Hang Sheng; Jianjian Shi; Xiaobing Shi; Ying-Hong Shi; Kahori Shiba-Fukushima; Jeng-Jer Shieh; Yohta Shimada; Shigeomi Shimizu; Makoto Shimozawa; Takahiro Shintani; Christopher J Shoemaker; Shahla Shojaei; Ikuo Shoji; Bhupendra V Shravage; Viji Shridhar; Chih-Wen Shu; Hong-Bing Shu; Ke Shui; Arvind K Shukla; Timothy E Shutt; Valentina Sica; Aleem Siddiqui; Amanda Sierra; Virginia Sierra-Torre; Santiago Signorelli; Payel Sil; Bruno J de Andrade Silva; Johnatas D Silva; Eduardo Silva-Pavez; Sandrine Silvente-Poirot; Rachel E Simmonds; Anna Katharina Simon; Hans-Uwe Simon; Matias Simons; Anurag Singh; Lalit P Singh; Rajat Singh; Shivendra V Singh; Shrawan K Singh; Sudha B Singh; Sunaina Singh; Surinder Pal Singh; Debasish Sinha; Rohit Anthony Sinha; Sangita Sinha; Agnieszka Sirko; Kapil Sirohi; Efthimios L Sivridis; Panagiotis Skendros; Aleksandra Skirycz; Iva Slaninová; Soraya S Smaili; Andrei Smertenko; Matthew D Smith; Stefaan J Soenen; Eun Jung Sohn; Sophia P M Sok; Giancarlo Solaini; Thierry Soldati; Scott A Soleimanpour; Rosa M Soler; Alexei Solovchenko; Jason A Somarelli; Avinash Sonawane; Fuyong Song; Hyun Kyu Song; Ju-Xian Song; Kunhua Song; Zhiyin Song; Leandro R Soria; Maurizio Sorice; Alexander A Soukas; Sandra-Fausia Soukup; Diana Sousa; Nadia Sousa; Paul A Spagnuolo; Stephen A Spector; M M Srinivas Bharath; Daret St Clair; Venturina Stagni; Leopoldo Staiano; Clint A Stalnecker; Metodi V Stankov; Peter B Stathopulos; Katja Stefan; Sven Marcel Stefan; Leonidas Stefanis; Joan S Steffan; Alexander Steinkasserer; Harald Stenmark; Jared Sterneckert; Craig Stevens; Veronika Stoka; Stephan Storch; Björn Stork; Flavie Strappazzon; Anne Marie Strohecker; Dwayne G Stupack; Huanxing Su; Ling-Yan Su; Longxiang Su; Ana M Suarez-Fontes; Carlos S Subauste; Selvakumar Subbian; Paula V Subirada; Ganapasam Sudhandiran; Carolyn M Sue; Xinbing Sui; Corey Summers; Guangchao Sun; Jun Sun; Kang Sun; Meng-Xiang Sun; Qiming Sun; Yi Sun; Zhongjie Sun; Karen K S Sunahara; Eva Sundberg; Katalin Susztak; Peter Sutovsky; Hidekazu Suzuki; Gary Sweeney; J David Symons; Stephen Cho Wing Sze; Nathaniel J Szewczyk; Anna Tabęcka-Łonczynska; Claudio Tabolacci; Frank Tacke; Heinrich Taegtmeyer; Marco Tafani; Mitsuo Tagaya; Haoran Tai; Stephen W G Tait; Yoshinori Takahashi; Szabolcs Takats; Priti Talwar; Chit Tam; Shing Yau Tam; Davide Tampellini; Atsushi Tamura; Chong Teik Tan; Eng-King Tan; Ya-Qin Tan; Masaki Tanaka; Motomasa Tanaka; Daolin Tang; Jingfeng Tang; Tie-Shan Tang; Isei Tanida; Zhipeng Tao; Mohammed Taouis; Lars Tatenhorst; Nektarios Tavernarakis; Allen Taylor; Gregory A Taylor; Joan M Taylor; Elena Tchetina; Andrew R Tee; Irmgard Tegeder; David Teis; Natercia Teixeira; Fatima Teixeira-Clerc; Kumsal A Tekirdag; Tewin Tencomnao; Sandra Tenreiro; Alexei V Tepikin; Pilar S Testillano; Gianluca Tettamanti; Pierre-Louis Tharaux; Kathrin Thedieck; Arvind A Thekkinghat; Stefano Thellung; Josephine W Thinwa; V P Thirumalaikumar; Sufi Mary Thomas; Paul G Thomes; Andrew Thorburn; Lipi Thukral; Thomas Thum; Michael Thumm; Ling Tian; Ales Tichy; Andreas Till; Vincent Timmerman; Vladimir I Titorenko; Sokol V Todi; Krassimira Todorova; Janne M Toivonen; Luana Tomaipitinca; Dhanendra Tomar; Cristina Tomas-Zapico; Sergej Tomić; Benjamin Chun-Kit Tong; Chao Tong; Xin Tong; Sharon A Tooze; Maria L Torgersen; Satoru Torii; Liliana Torres-López; Alicia Torriglia; Christina G Towers; Roberto Towns; Shinya Toyokuni; Vladimir Trajkovic; Donatella Tramontano; Quynh-Giao Tran; Leonardo H Travassos; Charles B Trelford; Shirley Tremel; Ioannis P Trougakos; Betty P Tsao; Mario P Tschan; Hung-Fat Tse; Tak Fu Tse; Hitoshi Tsugawa; Andrey S Tsvetkov; David A Tumbarello; Yasin Tumtas; María J Tuñón; Sandra Turcotte; Boris Turk; Vito Turk; Bradley J Turner; Richard I Tuxworth; Jessica K Tyler; Elena V Tyutereva; Yasuo Uchiyama; Aslihan Ugun-Klusek; Holm H Uhlig; Marzena Ułamek-Kozioł; Ilya V Ulasov; Midori Umekawa; Christian Ungermann; Rei Unno; Sylvie Urbe; Elisabet Uribe-Carretero; Suayib Üstün; Vladimir N Uversky; Thomas Vaccari; Maria I Vaccaro; Björn F Vahsen; Helin Vakifahmetoglu-Norberg; Rut Valdor; Maria J Valente; Ayelén Valko; Richard B Vallee; Angela M Valverde; Greet Van den Berghe; Stijn van der Veen; Luc Van Kaer; Jorg van Loosdregt; Sjoerd J L van Wijk; Wim Vandenberghe; Ilse Vanhorebeek; Marcos A Vannier-Santos; Nicola Vannini; M Cristina Vanrell; Chiara Vantaggiato; Gabriele Varano; Isabel Varela-Nieto; Máté Varga; M Helena Vasconcelos; Somya Vats; Demetrios G Vavvas; Ignacio Vega-Naredo; Silvia Vega-Rubin-de-Celis; Guillermo Velasco; Ariadna P Velázquez; Tibor Vellai; Edo Vellenga; Francesca Velotti; Mireille Verdier; Panayotis Verginis; Isabelle Vergne; Paul Verkade; Manish Verma; Patrik Verstreken; Tim Vervliet; Jörg Vervoorts; Alexandre T Vessoni; Victor M Victor; Michel Vidal; Chiara Vidoni; Otilia V Vieira; Richard D Vierstra; Sonia Viganó; Helena Vihinen; Vinoy Vijayan; Miquel Vila; Marçal Vilar; José M Villalba; Antonio Villalobo; Beatriz Villarejo-Zori; Francesc Villarroya; Joan Villarroya; Olivier Vincent; Cecile Vindis; Christophe Viret; Maria Teresa Viscomi; Dora Visnjic; Ilio Vitale; David J Vocadlo; Olga V Voitsekhovskaja; Cinzia Volonté; Mattia Volta; Marta Vomero; Clarissa Von Haefen; Marc A Vooijs; Wolfgang Voos; Ljubica Vucicevic; Richard Wade-Martins; Satoshi Waguri; Kenrick A Waite; Shuji Wakatsuki; David W Walker; Mark J Walker; Simon A Walker; Jochen Walter; Francisco G Wandosell; Bo Wang; Chao-Yung Wang; Chen Wang; Chenran Wang; Chenwei Wang; Cun-Yu Wang; Dong Wang; Fangyang Wang; Feng Wang; Fengming Wang; Guansong Wang; Han Wang; Hao Wang; Hexiang Wang; Hong-Gang Wang; Jianrong Wang; Jigang Wang; Jiou Wang; Jundong Wang; Kui Wang; Lianrong Wang; Liming Wang; Maggie Haitian Wang; Meiqing Wang; Nanbu Wang; Pengwei Wang; Peipei Wang; Ping Wang; Ping Wang; Qing Jun Wang; Qing Wang; Qing Kenneth Wang; Qiong A Wang; Wen-Tao Wang; Wuyang Wang; Xinnan Wang; Xuejun Wang; Yan Wang; Yanchang Wang; Yanzhuang Wang; Yen-Yun Wang; Yihua Wang; Yipeng Wang; Yu Wang; Yuqi Wang; Zhe Wang; Zhenyu Wang; Zhouguang Wang; Gary Warnes; Verena Warnsmann; Hirotaka Watada; Eizo Watanabe; Maxinne Watchon; Anna Wawrzyńska; Timothy E Weaver; Grzegorz Wegrzyn; Ann M Wehman; Huafeng Wei; Lei Wei; Taotao Wei; Yongjie Wei; Oliver H Weiergräber; Conrad C Weihl; Günther Weindl; Ralf Weiskirchen; Alan Wells; Runxia H Wen; Xin Wen; Antonia Werner; Beatrice Weykopf; Sally P Wheatley; J Lindsay Whitton; Alexander J Whitworth; Katarzyna Wiktorska; Manon E Wildenberg; Tom Wileman; Simon Wilkinson; Dieter Willbold; Brett Williams; Robin S B Williams; Roger L Williams; Peter R Williamson; Richard A Wilson; Beate Winner; Nathaniel J Winsor; Steven S Witkin; Harald Wodrich; Ute Woehlbier; Thomas Wollert; Esther Wong; Jack Ho Wong; Richard W Wong; Vincent Kam Wai Wong; W Wei-Lynn Wong; An-Guo Wu; Chengbiao Wu; Jian Wu; Junfang Wu; Kenneth K Wu; Min Wu; Shan-Ying Wu; Shengzhou Wu; Shu-Yan Wu; Shufang Wu; William K K Wu; Xiaohong Wu; Xiaoqing Wu; Yao-Wen Wu; Yihua Wu; Ramnik J Xavier; Hongguang Xia; Lixin Xia; Zhengyuan Xia; Ge Xiang; Jin Xiang; Mingliang Xiang; Wei Xiang; Bin Xiao; Guozhi Xiao; Hengyi Xiao; Hong-Tao Xiao; Jian Xiao; Lan Xiao; Shi Xiao; Yin Xiao; Baoming Xie; Chuan-Ming Xie; Min Xie; Yuxiang Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Congfeng Xu; En Xu; Haoxing Xu; Jing Xu; JinRong Xu; Liang Xu; Wen Wen Xu; Xiulong Xu; Yu Xue; Sokhna M S Yakhine-Diop; Masamitsu Yamaguchi; Osamu Yamaguchi; Ai Yamamoto; Shunhei Yamashina; Shengmin Yan; Shian-Jang Yan; Zhen Yan; Yasuo Yanagi; Chuanbin Yang; Dun-Sheng Yang; Huan Yang; Huang-Tian Yang; Hui Yang; Jin-Ming Yang; Jing Yang; Jingyu Yang; Ling Yang; Liu Yang; Ming Yang; Pei-Ming Yang; Qian Yang; Seungwon Yang; Shu Yang; Shun-Fa Yang; Wannian Yang; Wei Yuan Yang; Xiaoyong Yang; Xuesong Yang; Yi Yang; Ying Yang; Honghong Yao; Shenggen Yao; Xiaoqiang Yao; Yong-Gang Yao; Yong-Ming Yao; Takahiro Yasui; Meysam Yazdankhah; Paul M Yen; Cong Yi; Xiao-Ming Yin; Yanhai Yin; Zhangyuan Yin; Ziyi Yin; Meidan Ying; Zheng Ying; Calvin K Yip; Stephanie Pei Tung Yiu; Young H Yoo; Kiyotsugu Yoshida; Saori R Yoshii; Tamotsu Yoshimori; Bahman Yousefi; Boxuan Yu; Haiyang Yu; Jun Yu; Jun Yu; Li Yu; Ming-Lung Yu; Seong-Woon Yu; Victor C Yu; W Haung Yu; Zhengping Yu; Zhou Yu; Junying Yuan; Ling-Qing Yuan; Shilin Yuan; Shyng-Shiou F Yuan; Yanggang Yuan; Zengqiang Yuan; Jianbo Yue; Zhenyu Yue; Jeanho Yun; Raymond L Yung; David N Zacks; Gabriele Zaffagnini; Vanessa O Zambelli; Isabella Zanella; Qun S Zang; Sara Zanivan; Silvia Zappavigna; Pilar Zaragoza; Konstantinos S Zarbalis; Amir Zarebkohan; Amira Zarrouk; Scott O Zeitlin; Jialiu Zeng; Ju-Deng Zeng; Eva Žerovnik; Lixuan Zhan; Bin Zhang; Donna D Zhang; Hanlin Zhang; Hong Zhang; Hong Zhang; Honghe Zhang; Huafeng Zhang; Huaye Zhang; Hui Zhang; Hui-Ling Zhang; Jianbin Zhang; Jianhua Zhang; Jing-Pu Zhang; Kalin Y B Zhang; Leshuai W Zhang; Lin Zhang; Lisheng Zhang; Lu Zhang; Luoying Zhang; Menghuan Zhang; Peng Zhang; Sheng Zhang; Wei Zhang; Xiangnan Zhang; Xiao-Wei Zhang; Xiaolei Zhang; Xiaoyan Zhang; Xin Zhang; Xinxin Zhang; Xu Dong Zhang; Yang Zhang; Yanjin Zhang; Yi Zhang; Ying-Dong Zhang; Yingmei Zhang; Yuan-Yuan Zhang; Yuchen Zhang; Zhe Zhang; Zhengguang Zhang; Zhibing Zhang; Zhihai Zhang; Zhiyong Zhang; Zili Zhang; Haobin Zhao; Lei Zhao; Shuang Zhao; Tongbiao Zhao; Xiao-Fan Zhao; Ying Zhao; Yongchao Zhao; Yongliang Zhao; Yuting Zhao; Guoping Zheng; Kai Zheng; Ling Zheng; Shizhong Zheng; Xi-Long Zheng; Yi Zheng; Zu-Guo Zheng; Boris Zhivotovsky; Qing Zhong; Ao Zhou; Ben Zhou; Cefan Zhou; Gang Zhou; Hao Zhou; Hong Zhou; Hongbo Zhou; Jie Zhou; Jing Zhou; Jing Zhou; Jiyong Zhou; Kailiang Zhou; Rongjia Zhou; Xu-Jie Zhou; Yanshuang Zhou; Yinghong Zhou; Yubin Zhou; Zheng-Yu Zhou; Zhou Zhou; Binglin Zhu; Changlian Zhu; Guo-Qing Zhu; Haining Zhu; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Yanping Zhu; Yushan Zhu; Haixia Zhuang; Xiaohong Zhuang; Katarzyna Zientara-Rytter; Christine M Zimmermann; Elena Ziviani; Teresa Zoladek; Wei-Xing Zong; Dmitry B Zorov; Antonio Zorzano; Weiping Zou; Zhen Zou; Zhengzhi Zou; Steven Zuryn; Werner Zwerschke; Beate Brand-Saberi; X Charlie Dong; Chandra Shekar Kenchappa; Zuguo Li; Yong Lin; Shigeru Oshima; Yueguang Rong; Judith C Sluimer; Christina L Stallings; Chun-Kit Tong Journal: Autophagy Date: 2021-02-08 Impact factor: 13.391
Authors: Karol Cieminski; Damian Jozef Flis; Katarzyna Dzik; Jan Jacek Kaczor; Emilia Czyrko; Malgorzata Halon-Golabek; Mariusz Roman Wieckowski; Jedrzej Antosiewicz; Wieslaw Ziolkowski Journal: Sci Rep Date: 2021-10-22 Impact factor: 4.379
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8