Xiulong Xu1,2,3,4, Jing Sun1,2, Ruilong Song5, Michelle E Doscas3, Ashley J Williamson6, Jingsong Zhou7, Jun Sun8, Xinan Jiao4,9, Xiufan Liu9,10, Yi Li11. 1. Institute of Comparative Medicine, Yangzhou University, Yangzhou 225009, Jiangsu Province, P. R. China. 2. College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, Jiangsu Province, P. R. China. 3. Department of Anatomy and Cell Biology Rush University Medical Center, Chicago, IL 60612, USA. 4. Jiangsu Key Laboratory of Zoonosis, Yangzhou University Yangzhou 225009, Jiangsu Province, China. 5. Core Facility, Yangzhou University, Yangzhou 225009, Jiangsu Province, P. R. China. 6. Rush Medical College, Chicago, IL 60612, USA. 7. Department of Physiology, Kansas City University of Medicine and Biosciences, Kansas City, MO 64106, USA. 8. Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. 9. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou 225009, Jiangsu Province, China. 10. Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou 225009, China. 11. Lester and Sue Smith Breast Center, Baylor College of Medicine, Houston, TX 77030, USA.
Abstract
mTOR activation suppresses autophagy by phosphorylating ULK1 at S757 and suppressing its enzymatic activity. Here we report that feedback activation of mTOR in the PI-3 kinase pathway by two p70 S6 kinase (S6K1) inhibitors (PF-4708671 and A77 1726, the active metabolite of an immunosuppressive drug leflunomide) or by S6K1 knockdown did not suppress but rather induced autophagy. Suppression of S6K1 activity led to the phosphorylation and activation of AMPK, which then phosphorylated ULK1 at S555. While mTOR feedback activation led to increased phosphorylation of ULK1 at S757, this modification did not the disrupt ULK1-AMPK interaction nor dampen ULK1 S555 phosphorylation and the induction of autophagy. In addition, inhibition of S6K1 activity led to JNK activation, which also contributed to autophagy. 5Z-7-oxozeaenol, a specific inhibitor of TAK1, or TAK1 siRNA blocked A77 1726-induced activation of AMPK and JNK, and LC3 lipidation. Taken together, our study establishes S6K1 as a key player in the PI-3 kinase pathway to suppress autophagy through inhibiting AMPK and JNK in a TAK1-dependent manner.
mTOR activation suppresses autophagy by phosphorylating ULK1 at S757 and suppressing its enzymatic activity. Here we report that feedback activation of mTOR in the PI-3 kinase pathway by two p70 S6 kinase (S6K1) inhibitors (PF-4708671 and A77 1726, the active metabolite of an immunosuppressive drug leflunomide) or by S6K1 knockdown did not suppress but rather induced autophagy. Suppression of S6K1 activity led to the phosphorylation and activation of AMPK, which then phosphorylated ULK1 at S555. While mTOR feedback activation led to increased phosphorylation of ULK1 at S757, this modification did not the disrupt ULK1-AMPK interaction nor dampen ULK1 S555 phosphorylation and the induction of autophagy. In addition, inhibition of S6K1 activity led to JNK activation, which also contributed to autophagy. 5Z-7-oxozeaenol, a specific inhibitor of TAK1, or TAK1 siRNA blocked A77 1726-induced activation of AMPK and JNK, and LC3 lipidation. Taken together, our study establishes S6K1 as a key player in the PI-3 kinase pathway to suppress autophagy through inhibiting AMPK and JNK in a TAK1-dependent manner.
Macroautophagy (referred as autophagy hereafter) is a highly conserved catabolic process characterized by the formation of the double-membraned vesicles (autophagosomes), fusion with lysosomes, and degradation of cellular materials. Autophagy is activated primarily by nutrient and energy stress. Other autophagy inducers include hypoxia, anticancer drugs, damaged organelles, protein aggregates, and infectious agents. AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) sense energy stress and nutrient depletion, respectively, and play pivotal roles in regulating autophagy [1, 2]. AMPK directly phosphorylates ULK1 at S555 and activates it or indirectly activates ULK1 by inhibiting mTORC1 activity [3-5]. mTOR, a serine/threonine kinase that interacts with several adaptor proteins to form the mTOR complex 1 (mTORC1), phosphorylates ULK1/2 at serine 757 (S757), disrupts its interaction with AMPK and prevents it from activating the autophagy pathway [6, 7]. Inactivation of mTORC1 by nutrient insufficiency or by rapamycin, an inhibitor of mTOR, induces autophagy [6, 7]. S6K1 is a serine/threonine kinase downstream of mTORC1. S6K1 plays important roles in cancer, diabetes, obesity, and ageing [8]. S6K1 depletion mimics the effect of diet restriction [9, 10]. Interestingly, AMPK activity is up-regulated in the skeletal muscle tissues and myotubes of S6K1-deficientmice due to elevated AMP/ATP ratios [9, 11]. Sch9, an equivalent of mammalianS6K1 in yeast, has been reported to inhibit autophagy [12, 13]. Whether mammalianS6K1 also suppresses autophagy is incompletely understood.TAK1 is a serine/threonine kinase that plays a crucial role in regulating cell survival, differentiation, apoptosis, and inflammatory responses. TAK1 is activated by IL-1 and TGF-β receptors, Toll-like receptors (TLR), CD40, and the B cell receptor [14-16]. TAK1 is involved in activating several intracellular kinases, including p38, JNK, and I-kappa B kinase complex (IKK) [17-20]. TAK1 plays a critical role in activating the tumor suppressor protein LKB1, and AMPK T172 phosphorylation is inhibited in TAK1-deficient embryos and in TAK1-deficient embryonic fibroblast cells [21]. Herrero-Martin et al. reported that TAK1 plays a critical role in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced AMPK activation [22]. Inokuchi-Shimizu et al. [23] 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 [23]. These observations strongly suggest that autophagy induced by mTOR suppression is in part mediated through TAK1. The underlying molecular mechanisms remain to be defined.A77 1726 is the active metabolite of leflunomide (Arava™), an anti-inflammatory drug primarily used for treating rheumatoid arthritis. Mechanistic studies revealed that A77 1726 and its parental drug, leflunomide, are capable of inhibiting tyrosine phosphorylation and pyrimidine nucleotide synthesis [24-31]. 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 PDGF receptor [24-28]. Recently, we reported that A77 1726 and its parental drug, leflunomide, inhibit the activity of S6K1 in an in vitro kinase assay and in cell culture, and that inhibition of S6K1 activity by A77 1726 leads to the feedback activation of the PI-3 kinase pathway [32]. Here we report that mTOR feedback activation by A77 1726 or PF-4708671 did not inhibit but rather induced autophagy. We also found that A77 1726-induced autophagy was mediated through inhibiting S6K1 activity, subsequently leading to activation of AMPK and JNK through TAK1, and that activation of AMPK and JNK both contributed to A77 1726-induced autophagy.
RESULTS
A77 1726 induces autophagy
Our recent study showed that A77 1726 suppresses S6K1 activity and subsequently induces feedback activation of PI3K, AKT, and mTOR in A375 cells [32]. Since mTOR activation suppresses autophagy [6], we tested if mTOR feedback activation by A77 1726 also suppressed autophagy. Unexpectedly, A77 1726 induced LC3-II lipidation in a dose-dependent manner in A375 (Figure 1A), MCF-7 breast cancer cells (Figure 1B), and C2C12 myotubes (Figure 1C). Rapamycin included as a positive control was less effective than A77 1726 to increase LC3-II levels in A375 cells (Figure 1A). Leflunomide, the parental drug of A77 1726, increased LC3-II levels too in A375 cells in a dose-dependent manner (Figure 1D). Increased LC3-II lipidation could be observed 8 hr after the addition of A77 1726 and lasted up to 48 hr in A375 cells (Figure 1E). Confocal microscopic fluorescence analysis revealed that LC3 formed autophagosomes in A375 cells in the presence of A77 1726, leflunomide, or rapamycin (Figure 2A). Enumeration of autophagosomes showed that A77 1726, leflunomide, and rapamycin all significantly increased the number of puncta (Figure 2B). Increased numbers of autophagosome puncta were also observed in MCF-7 cells treated with A77 1726, leflunomide, or rapamycin (data not shown). To determine if increased LC3-II lipidation was due to the stall of autophagy flux or was indeed due to the induction of autophagy, we tested the effect of bafilomycin and colchicine on A77 1726-induced autophagy. As shown in Figure 1F, A77 1726, bafilomycin or colchicine alone increased the levels of both LC3-I and LC3-II. Combination of A77 1726 with bafilomycin or colchicine further increased the ratio of LC3-II to LC-I, compared to bafilomycin or colchicine alone. These results suggest that A77 1726 induces autophagy, and that increased LC3-II levels are not due to the inhibition of the autophagy flux.
Figure 1
A77 1726 increases LC3-II expression
(A-C) Dose-dependent increase of LC3-II levels. A375 (A), MCF-7 (B), and C2C12 (C) cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 hr. Rapamycin (Rapa) (20 nM) was included as a control. LC3 and actin expression was analyzed by Western blot. (D) A375 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of leflunomide for 16 hr. LC3 and actin expression were analyzed by Western blot. (E) Time-dependent increase of LC3-II lipidation. A375 cells were incubated in the presence of A77 1726 (200 μM) for the indicated time. Cell lysates were analyzed for LC3 and actin levels by Western blot. (F) The effect of bafilomycin and colchicine. A375 cells seeded in 6-well plates were incubated in complete DMEM medium in the absence or presence of bafilomycin (10 or 40 nM) or colchicine (5 μM) for 16 hr. Cell lysates were analyzed for LC3 and actin expression by Western blot.
Figure 2
Induction of autophagosomes by A77 1726
A375 cells were transfected with the expression vector pmLC3-RFP. The cells were left untreated or treated with A77 1726 (200 μM), rapamycin (20 nM), or leflunomide (200 μM) for 16 hr. Autophagosomes were visualized under a confocal microscope (A). The puncta of autophagosomes were counted under a fluorescence microscope and plotted in a bar graph with statistical analysis (B). **p<0.01, compared to the control.
A77 1726 increases LC3-II expression
(A-C) Dose-dependent increase of LC3-II levels. A375 (A), MCF-7 (B), and C2C12 (C) cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 hr. Rapamycin (Rapa) (20 nM) was included as a control. LC3 and actin expression was analyzed by Western blot. (D) A375 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of leflunomide for 16 hr. LC3 and actin expression were analyzed by Western blot. (E) Time-dependent increase of LC3-II lipidation. A375 cells were incubated in the presence of A77 1726 (200 μM) for the indicated time. Cell lysates were analyzed for LC3 and actin levels by Western blot. (F) The effect of bafilomycin and colchicine. A375 cells seeded in 6-well plates were incubated in complete DMEM medium in the absence or presence of bafilomycin (10 or 40 nM) or colchicine (5 μM) for 16 hr. Cell lysates were analyzed for LC3 and actin expression by Western blot.
Induction of autophagosomes by A77 1726
A375 cells were transfected with the expression vector pmLC3-RFP. The cells were left untreated or treated with A77 1726 (200 μM), rapamycin (20 nM), or leflunomide (200 μM) for 16 hr. Autophagosomes were visualized under a confocal microscope (A). The puncta of autophagosomes were counted under a fluorescence microscope and plotted in a bar graph with statistical analysis (B). **p<0.01, compared to the control.As an inhibitor of DHO-DHase, A77 1726 inhibits pyrimidine nucleotide synthesis [33]. To determine if increased LC3-II lipidation was due to pyrimidine nucleotide depletion, we tested whether exogenous uridine blocked A77 1726-induced LC3-II lipidation. According to our previous studies, exogenous uridine added into rapidly proliferating cells or injected into mice can be readily uptaken by cells and normalize intracellular pyrimidine nucleotide levels [24, 26] Uridine (200 μM) itself had no effect on LC3-II levels and did not block A77 1726- (Figure 3A) or leflunomide-induced (Figure 3B) LC3 lipidation in A375 cells. Uridine had also no effect on A77 1726- or leflunomide-induced LC3-II lipidation in MCF-7 cells (Figure 3C). Moreover, brequinar sodium (BQR), a potent inhibitor of pyrimidine nucleotide synthesis, did not increase but rather slightly decreased LC3-II lipidation (Figure 3D).
Figure 3
A77 1726 increases LC3-II levels independent of pyrimidine nucleotide depletion and the feedback activation of the PI-3 and MAP kinase pathways
(A&B) A375 cells seeded in a 6-well plate were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) (A) or leflunomide (200 μM) (B) and/or uridine (200 μM) for 16 hr. Cells were harvested and analyzed for LC3 and actin expression by Western blot. (C) MCF7 cells were similarly treated as in A and B and analyzed for LC3 and actin levels. (D) The effect of brequinar sodium (BQR) on LC3 lipidation. A375 cells seeded in a 6-well plate were incubated in complete DMEM medium in the absence or presence of BQR (10 μM) for 16 or 24 hr. LC3 and actin levels were analyzed by Western blot. (E) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PLX4720 (1 μM) or U0126 (10 μM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for LC3 and actin expression. (F) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PPP (1 μM), LY294002 (10 μM) or rapamycin (20 nM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for LC3 and actin expression.
A77 1726 increases LC3-II levels independent of pyrimidine nucleotide depletion and the feedback activation of the PI-3 and MAP kinase pathways
(A&B) A375 cells seeded in a 6-well plate were incubated in complete DMEM medium in the absence or presence of A77 1726 (200 μM) (A) or leflunomide (200 μM) (B) and/or uridine (200 μM) for 16 hr. Cells were harvested and analyzed for LC3 and actin expression by Western blot. (C) MCF7 cells were similarly treated as in A and B and analyzed for LC3 and actin levels. (D) The effect of brequinar sodium (BQR) on LC3 lipidation. A375 cells seeded in a 6-well plate were incubated in complete DMEM medium in the absence or presence of BQR (10 μM) for 16 or 24 hr. LC3 and actin levels were analyzed by Western blot. (E) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PLX4720 (1 μM) or U0126 (10 μM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for LC3 and actin expression. (F) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PPP (1 μM), LY294002 (10 μM) or rapamycin (20 nM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for LC3 and actin expression.
A77 1726-induced autophagy is independent of the feedback activation of the PI-3 and MAP kinase pathways
Our recent study showed that A77 1726 induces the feedback activation of the PI-3 and MAP kinase pathways; and that PLX4720, an inhibitor of Raf kinase, and U0126, a MEK inhibitor, block A77 1726-induced phosphorylation of ERK1/2T202/Y204 and MEK1/2S217/S221 [32]. Here we found that these inhibitors did not change the levels of LC3-II lipidation in the presence of A77 1726 (Figure 3E). The IGF-1 receptor tyrosine kinase is responsible for S6K1-mediated negative feedback activation of the MAP and PI-3 kinase pathways [34]. As shown in Figure 3F, PPP, an inhibitor of IGF-1 receptor; LY294002, a PI-3 kinase inhibitor, and rapamycin, an inhibitor of mTOR, were unable to block A77 1726-induced LC3-II increase (Figure 3F).
Suppression of S6K1 activity induces autophagy
To investigate the role of S6K1 in mediating A77 1726-induced autophagy, we first examined the effect of S6K1 knockdown on autophagy in A375 cells. As shown in Figure 4A, S6K1 siRNA very effectively silenced S6K1 expression. S6K1 knockdown led to decreased S6 phosphorylation but increased the levels of LC3-II. Consistently, S6K1 knockdown also led to the increase of the number of LC3-RFP puncta (Figure 4C & 4E). Similar to A77 1726, PF-4708671, a specific inhibitor of S6K1, inhibited S6 phosphorylation but induced the feedback activation of the PI-3 kinase pathway, as shown by increased AKT and S6K1 phosphorylation (Figure 4B). Consistently, PF-4708671 induced LC3 lipidation in a dose-dependent manner in A375 cells (Figure 4B) and increased the number of LC3-RFP puncta (Figure 4D & 4F).
Figure 4
Role of S6K1 in autophagy
(A) The effect of S6K1 knockdown on LC3-II lipidation.A375 cells were transfected with scrambled or S6K1 siRNA (2.5 nmole each). After incubation for 48 hr, the cells were harvested and analyzed for S6K1 expression and phosphorylation of the indicated proteins by Western blot. (B) The effect of the S6K1 inhibitor on LC3-II expression.A375 cells seeded in a 6-well plate were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of PF-4708671 for 16 hr. Cells were harvested and analyzed for LC3 and actin expression by Western blot. (C) The effect of S6K1 knockdown on autophagosome formation. A375 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 hr, the cells were fixed in methanol and visualized for autophagosomes under a fluorescent microscope. (D) The effect of the S6K1 inhibitor on LC3-II expression.A375 cells seeded on coverslips were transfected with LC3-RFP expression vector. After incubation for 24 hr, the cells were treated with PF-4708671 (10 μM) for 16 hr. Cells were fixed in methanol and visualized for autophagosomes under a fluorescence microscope. (E & F) The puncta of autophagosomes were counted under a fluorescent microscope plotted in a bar graph with statistical analysis. **p<0.01, compared to the control.
Role of S6K1 in autophagy
(A) The effect of S6K1 knockdown on LC3-II lipidation.A375 cells were transfected with scrambled or S6K1 siRNA (2.5 nmole each). After incubation for 48 hr, the cells were harvested and analyzed for S6K1 expression and phosphorylation of the indicated proteins by Western blot. (B) The effect of the S6K1 inhibitor on LC3-II expression.A375 cells seeded in a 6-well plate were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of PF-4708671 for 16 hr. Cells were harvested and analyzed for LC3 and actin expression by Western blot. (C) The effect of S6K1 knockdown on autophagosome formation. A375 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 hr, the cells were fixed in methanol and visualized for autophagosomes under a fluorescent microscope. (D) The effect of the S6K1 inhibitor on LC3-II expression.A375 cells seeded on coverslips were transfected with LC3-RFP expression vector. After incubation for 24 hr, the cells were treated with PF-4708671 (10 μM) for 16 hr. Cells were fixed in methanol and visualized for autophagosomes under a fluorescence microscope. (E & F) The puncta of autophagosomes were counted under a fluorescent microscope plotted in a bar graph with statistical analysis. **p<0.01, compared to the control.
A77 1726 induces AMPK and ULK1 phosphorylation by inhibiting S6K1 activity
ULK1 is phosphorylated at S555 and activated by AMPK [3, 35]. Previous studies have shown that AMPK is activated in S6K1-deficient mice [9, 11]. A77 1726 may induce autophagy by inhibiting S6K1 activity and subsequent activation of AMPK and then ULK1. Indeed, we found that A77 1726 induced AMPK phosphorylation at T172 and ULK1 phosphorylation at S555 in A375 cells in a dose- (Figure 5A) and time-dependent (Figure 5B) manner. mTOR phosphorylates ULK1 at S757 and inhibits its activity as well as autophagy [6, 7]. mTOR is activated by A77 1726 due to the feedback activation of the PI-3 kinase pathway [32]. As expected, A77 1726 induced ULK1 phosphorylation at S757 in A375 cells in a dose- (Figure 5A) and time-dependent (Figure 5B) manner. Rapamycin had little effect on AMPKT172 and ULK1S555 phosphorylation but inhibited ULK1S757 phosphorylation (Figure 5A). Consistently, A77 1726 and leflunomide induced AMPKT172 and ULK1S555 phosphorylation in C2C12 cells in a dose-dependent manner (Figure 5C & 5D). ULK1 S757 phosphorylation disrupts AMPK and ULK1 interaction [6]. We tested whether mTOR feedback activation and ULK1 S757 phosphorylation by A77 1726 inhibited the formation of the AMPK-ULK complex. As shown in Figure 5E, the levels of ULK1 in anti-AMPK immunoprecipitate of A77 1726-treated A375 cells were equal to those in the untreated control. In contrast, inhibition of ULK1 S757 phosphorylation by rapamycin led to increased AMPK and ULK1 interaction (Figure 5E).
Figure 5
A77 1726 induces AMPK and ULK1 phosphorylation
(A & B) Time- and dose-dependent induction of AMPK and ULK1 phosphorylation by A77 1726. A375 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 hr (A) or in the presence of A77 1726 (200 μM) for the indicated time (B). (C & D) Dose-dependent induction of AMPK and ULK1 phosphorylation by A77 1726 (C) or leflunomide (D) in C2C12 myotubes. C2C12 cells were seeded in 6-well plates and differentiated into myotubes. Myotubes were treated with the indicated concentrations of A77 1726 or leflunomide for 16 hr. Cell lysates were prepared and analyzed by Western blot with the indicated antibodies. (E) A77 1726 does not disrupt AMPK and ULK1 interaction. A375 cells were incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (20 nM) for 16 hr. Cell lysates were immunoprecipitated with an anti-ULK1 antibody, followed by Western blot analysis with an anti-ULK1 or anti-AMPK antibody. Unimmunoprecipitated cell lysates were included as input controls.
A77 1726 induces AMPK and ULK1 phosphorylation
(A & B) Time- and dose-dependent induction of AMPK and ULK1 phosphorylation by A77 1726. A375 cells were incubated in complete DMEM medium in the absence or presence of the indicated concentrations of A77 1726 for 16 hr (A) or in the presence of A77 1726 (200 μM) for the indicated time (B). (C & D) Dose-dependent induction of AMPK and ULK1 phosphorylation by A77 1726 (C) or leflunomide (D) in C2C12 myotubes. C2C12 cells were seeded in 6-well plates and differentiated into myotubes. Myotubes were treated with the indicated concentrations of A77 1726 or leflunomide for 16 hr. Cell lysates were prepared and analyzed by Western blot with the indicated antibodies. (E) A77 1726 does not disrupt AMPK and ULK1 interaction. A375 cells were incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (20 nM) for 16 hr. Cell lysates were immunoprecipitated with an anti-ULK1 antibody, followed by Western blot analysis with an anti-ULK1 or anti-AMPK antibody. Unimmunoprecipitated cell lysates were included as input controls.
The effect of the feedback activation of the PI-3 and MAP kinase pathways on A77 1726-induced AMPK and ULK1 phosphorylation
We first determined the effect of the feedback activation of the PI-3 kinase pathway on AMPK and ULK1 phosphorylation. As shown in Figure 6A, all three inhibitors of the PI-3 kinase pathway, including the IGF-1 receptor (PPP), PI-3 kinase (LY294002), and mTOR (rapamycin), did not significantly block A77 1726-induced AMPK phosphorylation. All three inhibitors had little effect on A77 1726 induced ULK1 phosphorylation at S555. In contrast, all three inhibitors almost completely inhibited A77 1726-induced ULK1 phosphorylation at S757. These observations confirmed that increased ULK1 phosphorylation at S757 is indeed mediated by feedback activation of the PI-3 kinase pathway through mTOR. We next determined if feedback activation of the MAP kinase pathway was involved in A77 1726-induced AMPK and ULK1 phosphorylation. As shown in Figure 6B, PLX4720, an inhibitor of B-Raf kinase, and U0126, an inhibitor of MEK kinase, had little or no effect in A77 1726-induced AMPK and ULK1 phosphorylation at both S555 and S757.
Figure 6
A77 1726-induced AMPK and ULK1 phosphorylation is independent of the feedback activation of the PI-3 and MAP kinase pathways
(A) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PPP (1 μM), LY294002 (10 μM), or rapamycin (20 nM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for AMPK and ULK1 phosphorylation. (B) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PLX4720 (1 μM), U0126 (10 μM), or PD98059 (10 μM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for ULK and AMPK phosphorylation with indicated antibodies.
A77 1726-induced AMPK and ULK1 phosphorylation is independent of the feedback activation of the PI-3 and MAP kinase pathways
(A) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PPP (1 μM), LY294002 (10 μM), or rapamycin (20 nM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for AMPK and ULK1 phosphorylation. (B) A375 cells seeded in 6-well plates were pre-treated with vehicle (0.1% dimethyl sulfoxide), PLX4720 (1 μM), U0126 (10 μM), or PD98059 (10 μM) for 1 hr, followed by addition of A77 1726 (200 μM) and incubation for 16 hr. Cells were harvested and analyzed for ULK and AMPK phosphorylation with indicated antibodies.
Role of S6K1 in regulating AMPK and ULK1 phosphorylation
To confirm that A77 1726-induced AMPK and ULK1 phosphorylation was mediated by inhibiting S6K1 activity, we tested if S6K1 knockdown or inhibition of S6K1 activity by PF-4708671 also led to increased AMPK and ULK1 phosphorylation. As shown in Figure 7A, suppression of S6K1 expression led to increased AMPK and ULK1 phosphorylation at S555 and S757. PF-470867 induced AMPK phosphorylation in a dose-dependent manner (Figure 7B). Due to its strong ability to induce the feedback activation of the PI-3 kinase pathway, PF-470867 induced ULK1 phosphorylation at S555 and S757 even at low concentrations (Figure 7B). These results collectively suggest that inhibition of S6K1 activity plays a critical role in inducing AMPK and ULK1 phosphorylation.
Figure 7
The role of AMPK in A77 1726-induced autophagy
(A & B) S6K1 knockdown and PF-4708671 induces AMPK and ULK1 phosphorylation. (A) A375 cells were transfected with S6K1 siRNA. After incubation for 48 hr, the cells were harvested and analyzed for AMPK and ULK1 phosphorylation with indicated antibodies. (B) A375 cells were treated with the indicated concentrations of PF-4708671 for 16 hr and analyzed for AMPK and ULK1 phosphorylation. (C) Inhibition of AMPK and ULK1 phosphorylation and LC3 expression by compound C (CC). A375 cells were incubated in the absence or presence of A77 1726 (200 μM) and/or CC (1 μM) for 16 hr. Cell lysates were prepared and analyzed for ULK and AMPK phosphorylation, and for LC3 and actin expression by Western blot. (D) The effect of AMPK activators on AMPK and ULK1 phosphorylation and LC3 expression. A375 cells were incubated in the absence or presence of A77 1726 (200 μM), oligomycin (10 μM) or metformin (5 mM) for 16 hr. Cell lysates were prepared and analyzed by Western blot with the indicated antibodies.
The role of AMPK in A77 1726-induced autophagy
(A & B) S6K1 knockdown and PF-4708671 induces AMPK and ULK1 phosphorylation. (A) A375 cells were transfected with S6K1 siRNA. After incubation for 48 hr, the cells were harvested and analyzed for AMPK and ULK1 phosphorylation with indicated antibodies. (B) A375 cells were treated with the indicated concentrations of PF-4708671 for 16 hr and analyzed for AMPK and ULK1 phosphorylation. (C) Inhibition of AMPK and ULK1 phosphorylation and LC3 expression by compound C (CC). A375 cells were incubated in the absence or presence of A77 1726 (200 μM) and/or CC (1 μM) for 16 hr. Cell lysates were prepared and analyzed for ULK and AMPK phosphorylation, and for LC3 and actin expression by Western blot. (D) The effect of AMPK activators on AMPK and ULK1 phosphorylation and LC3 expression. A375 cells were incubated in the absence or presence of A77 1726 (200 μM), oligomycin (10 μM) or metformin (5 mM) for 16 hr. Cell lysates were prepared and analyzed by Western blot with the indicated antibodies.
AMPK mediates A77 1726-induced ULK1 phosphorylation and autophagy
To confirm that the effect of A77 1726 on ULK1 phosphorylation was indeed mediated through AMPK, we first tested if compound C (CC), an inhibitor of AMPK, was able to block A77 1726-induced ULK1 phosphorylation and LC3 expression. As shown in Figure 7C, CC had no effect on A77 1726-induced AMPK phosphorylation but blocked A77 1726-induced ULK1 phosphorylation at ULK1 S555 and LC3-II lipidation. CC blocked A77 1726-induced ULK1 S757 phosphorylation, probably as the result of inhibition of raptor phosphorylation and inhibition of mTOR activity [36]. In contrast, two AMPK activators, oligomycin and metformin, induced AMPK and ULK1 S555 phosphorylation (Figure 7D). Both oligomycin and metformin induced LC3 lipidation and slightly increased ULK1 S757 phosphorylation. These observations collectively suggest that A77 1726 induces autophagy by AMPK activation-induced ULK1 phosphorylation.
A77 1726 induces p62 expression
p62 is a ubiquitin-binding protein that sequesters ubiquitinated proteins for lysosomal degradation through interacting with LC3-II in autophagosomes [37]. The p62 level is usually decreased following autophagy induction. However, p62 expression is induced by resveratrol, an autophagy inducer, by transcriptional up-regulation through the MAP kinase-activated AP1 transcription factor [38]. As shown in Figure 8A, A77 1726 increased p62 levels after a prolonged exposure for 24 hr. Rapamycin slightly induced p62 expression. A77 1726-induced p62 expression was time-dependent (Figure 8B). Induction of p62 by A77 1726 or leflunomide was not due to its effect on pyrimidine nucleotide synthesis, since exogenous uridine was unable to block A77 1726- or leflunomide-induced p62 expression (Figure 8C). The inhibitors of the MAP kinase pathway, including PLX4720, U0126, and PD98059, had no effect on A77 1726-induced p62 expression (Figure 8D). The inhibitors of the PI-3 kinase pathway, including PPP, LY294002, and rapamycin, did not increase A77 1726-induced p62 expression (Figure 8E).
Figure 8
A77 1726 induces p62 expression
(A & B) Dose- and time-dependent induction of p62 by A77 1726.A375 cells were treated with the indicated concentration of A77 1726 or rapamycin (20 nM) (A) for 16 hr or treated with A77 1726 (200 μM) for the indicated time (B). Cell lysates were prepared and analyzed for p62 and actin expression. (C-E) A375 cells were treated with A77 1726 (200 μM) or leflunomide in the absence or presence of uridine (C), MAP kinase pathway inhibitors (D), or the PI-3 kinase inhibitors (E) for 16 hr. Cells were harvested and analyzed for p62 and actin levels by Western blot.
A77 1726 induces p62 expression
(A & B) Dose- and time-dependent induction of p62 by A77 1726.A375 cells were treated with the indicated concentration of A77 1726 or rapamycin (20 nM) (A) for 16 hr or treated with A77 1726 (200 μM) for the indicated time (B). Cell lysates were prepared and analyzed for p62 and actin expression. (C-E) A375 cells were treated with A77 1726 (200 μM) or leflunomide in the absence or presence of uridine (C), MAP kinase pathway inhibitors (D), or the PI-3 kinase inhibitors (E) for 16 hr. Cells were harvested and analyzed for p62 and actin levels by Western blot.
JNK activation is required for A77 1726-induced p62 expression
An earlier study showed that activation of JNK is required for the increased p62 expression by resveratrol in colorectal cancer cell lines [38, 39]. Here we tested whether A77 1726 was able to activate JNK, subsequently leading to increased p62 expression. A77 1726 induced JNK and Jun phosphorylation in a time- and dose-dependent manner (Figure 9A & 9B). SP600125, a specific inhibitor of JNK, blocked A77 1726-induced Jun and JNK phosphorylation. SP600125 also blocked p62 expression and LC3-II lipidation but had no effect on A77 1726-induced AMPK and ULK1 phosphorylation (Figure 9C). These results suggest that JNK activation is responsible for A77 1726-induced p62 expression.
Figure 9
JNK activation is required for A77 1726-induced p62 expression
A375 cells were treated with the indicated concentration of A77 1726 or rapamycin (20 nM) (A) for 16 hr or treated with A77 1726 (200 μM) for the indicated time (B). (C) A375 cells were treated in the absence or presence of A77 1726 (200 μM) and/or SP6000125 (10 μM) for 16 hr. Cell lysates were prepared and analyzed for the protein levels with the indicated antibodies.
JNK activation is required for A77 1726-induced p62 expression
A375 cells were treated with the indicated concentration of A77 1726 or rapamycin (20 nM) (A) for 16 hr or treated with A77 1726 (200 μM) for the indicated time (B). (C) A375 cells were treated in the absence or presence of A77 1726 (200 μM) and/or SP6000125 (10 μM) for 16 hr. Cell lysates were prepared and analyzed for the protein levels with the indicated antibodies.The inability of SP600125 to block A77 1726-induced AMPK and ULK1 phosphorylation suggests that AMPK is not involved in A77 1726-induced JNK activation. As shown in Figure 10A, oligomycin and metformin, two AMPK activators, had no or little effect on JNK and Jun phosphorylation nor induced p62 expression. Compound C, an AMPK inhibitor, was unable to block A77 1726-induced JNK and Jun phosphorylation as well as p62 expression (Figure 10B). S6K1 knockdown led to increased JNK and Jun phosphorylation as well as increased p62 expression (Figure 10C). Consistently, PF-4708671 induced JNK and Jun phosphorylation as well as p62 expression in a dose-dependent manner (Figure 10D). These observations collectively suggest that JNK activation by A77 1726 is mediated by inhibition of S6K1 activity.
Figure 10
JNK activation is required for A77 1726-induced p62 expression
(A) The effect of AMPK activators on JNK activation. A375 cells were incubated in the absence or presence of A77 1726 (200 μM), oligomycin (10 μM), or metformin (5 mM) for 16 hr. Cells were harvested and analyzed with the antibodies against phospho-JNK, phospho-Jun, p62 and actin. (B) A375 cells were incubated in the absence or presence of A77 1726 (200 μM) and/or compound C (CC) (1 μM) for 16 hr. Cell lysates were prepared and analyzed for ULK and AMPK phosphorylation, and for LC3 and actin expression by Western blot. (C) A375 cells were transfected with S6K1 siRNA. After incubation for 48 hr, the cells were harvested and analyzed for Jun, JNK, p62, and actin expression. (D) A375 cells were treated with the indicated concentrations of PF-4708671 for 16 hr. Cells were harvested and analyzed for Jun, JNK, p62, and actin expression.
(A) The effect of AMPK activators on JNK activation. A375 cells were incubated in the absence or presence of A77 1726 (200 μM), oligomycin (10 μM), or metformin (5 mM) for 16 hr. Cells were harvested and analyzed with the antibodies against phospho-JNK, phospho-Jun, p62 and actin. (B) A375 cells were incubated in the absence or presence of A77 1726 (200 μM) and/or compound C (CC) (1 μM) for 16 hr. Cell lysates were prepared and analyzed for ULK and AMPK phosphorylation, and for LC3 and actin expression by Western blot. (C) A375 cells were transfected with S6K1 siRNA. After incubation for 48 hr, the cells were harvested and analyzed for Jun, JNK, p62, and actin expression. (D) A375 cells were treated with the indicated concentrations of PF-4708671 for 16 hr. Cells were harvested and analyzed for Jun, JNK, p62, and actin expression.
Role of TAK1 in S6K1-mediated regulation of autophagy
S6K1 negatively regulates the activity of TAK1 [40], a serine/threonine kinase that activates JNK [17-20]. TAK1 also phosphorylates and activates LKB1 [21], a tumor suppressor responsible for AMPK T172 autophosphorylation and activation. Here we tested if S6K1 suppression A77 1726 led to the activation of TAK1, subsequently activating AMPK and JNK. As shown in Figure 11A, A77 1726 induced TAK1 phosphorylation in a time- and dose-dependent manner. A77 1726 treatment also led to the presence of multiple bands, probably as a result of ubiquitination (Figure 11A). 5Z-7-oxozeaenol, an inhibitor of TAK1, blocked A77 1726-induced phosphorylation of AMPK, ULK S555, JNK, and Jun phosphorylation, and blocked A77 1726-induced LC3 lipidation (Figure 11B). TAK1 siRNA had similar effect on A77 1726-induced protein phosphorylation and LC3 lipidation (Figure 11C). Co-immunoprecipitation revealed that S6K1 interacted with TAK1, whereas A77 1726 and rapamycin did not increase interaction of these two kinases (Figure 11D). These observations suggest that TAK1 plays a critical role in mediating A77 1726-induced activation of AMPK and JNK.
Figure 11
TAK1 mediates A77 1726-induced AMPK and JNK activation
(A) A77 1726 induces TAK1 phosphorylation. A375 cells were incubated in the absence or presence of the indicated concentrations of A77 1726 or rapamycin (20 nM) or A77 1726 (200 μM) for the indicated time. Cell lysates were analyzed with an anti-TAK1T184/187 phosphorylation antibody, followed by re-probing with an anti-TAK1 antibody. (B) The effect of the TAK1 inhibitor on protein phosphorylation and LC3-II levels.A375 cells were treated with A77 1726 (200 μM) and/or 5Z (5 μM) for 16 hr. Cell lysates were analyzed for the phosphorylation of indicated proteins by Western blot. (C) The effect of TAK1 knockdown on protein phosphorylation and LC3-II levels.A375 cells were transfected with scrambled or TAK1 siRNA (2.5 nmole each). After incubation for 48 hr, the cells were left untreated or treated with A77 1726 for 16 hr. Cells were harvested and analyzed for the indicated proteins by Western blot. (D) S6K1 interacts with TAK1. A375 cells seeded in a 6-well plate were incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (20 nM) for 16 hr. Cell lysates were immunoprecipitated with an anti-TAK1 or anti-S6K1 antibody, followed by Western blot analysis with an anti-TAK1 and anti-S6K1 antibodies. Unimmunoprecipitated cell lysates were included as input controls.
TAK1 mediates A77 1726-induced AMPK and JNK activation
(A) A77 1726 induces TAK1 phosphorylation. A375 cells were incubated in the absence or presence of the indicated concentrations of A77 1726 or rapamycin (20 nM) or A77 1726 (200 μM) for the indicated time. Cell lysates were analyzed with an anti-TAK1T184/187 phosphorylation antibody, followed by re-probing with an anti-TAK1 antibody. (B) The effect of the TAK1 inhibitor on protein phosphorylation and LC3-II levels.A375 cells were treated with A77 1726 (200 μM) and/or 5Z (5 μM) for 16 hr. Cell lysates were analyzed for the phosphorylation of indicated proteins by Western blot. (C) The effect of TAK1 knockdown on protein phosphorylation and LC3-II levels.A375 cells were transfected with scrambled or TAK1 siRNA (2.5 nmole each). After incubation for 48 hr, the cells were left untreated or treated with A77 1726 for 16 hr. Cells were harvested and analyzed for the indicated proteins by Western blot. (D) S6K1 interacts with TAK1. A375 cells seeded in a 6-well plate were incubated in the absence or presence of A77 1726 (200 μM) or rapamycin (20 nM) for 16 hr. Cell lysates were immunoprecipitated with an anti-TAK1 or anti-S6K1 antibody, followed by Western blot analysis with an anti-TAK1 and anti-S6K1 antibodies. Unimmunoprecipitated cell lysates were included as input controls.
DISCUSSION
Leflunomide is a drug with multiple therapeutic potentials, including immunosuppressive, anti-viral and anti-cancer activities [41-43]. Our recent study showed that A77 1726 is an inhibitor of S6K1, and that inhibition of S6K1 activity leads to the feedback activation of the PI-3 kinase pathway [32]. Our present study provides several lines of evidence that A77 1726 was capable of inducing autophagy: 1) A77 1726 increased the levels of LC3-II and the ratio of LC3-II to LC3-I (Figure 1); 2) A77 1726 induced the accumulation of autophagosome puncta (Figure 2); 3) bafilomycin and colchicine increased A77 1726-induced LC3-II expression (Figure 1); 4) AMPK T172 and ULK1 S555 phosphorylation was increased in A77 1726-treated cells (Figure 5). These observations collectively suggest that A77 1726 induces autophagy.A77 1726 inhibits the activity of at least three types of enzymes: DHO-DHase, protein tyrosine kinases, and S6K1 [26, 27, 32]. Exogenous uridine was unable to block A77 1726-induced LC3-II levels and autophagosome formation, suggesting that A77 1726-induced autophagy is independent of its inhibitory effect on pyrimidine nucleotide synthesis. In support of this notion, brequinar sodium, a much stronger inhibitor of DHO-DHase than leflunomide, was unable to induce LC3-II lipidation (Figure 3D). Activation of AMPK plays a critical role in inducing autophagy [44]. S6K1 knockdown or inhibition of S6K1 activity by PF-4708671 led to AMPK and ULK1 phosphorylation and activation (Figure 7), and subsequent to autophagy (Figure 4). A77 1726 is an inhibitor of S6K1 activity in an in vitro kinase assay and in cell culture [32]. AMPK T172 and ULK1 S555 phosphorylation was increased in A77 1726-treated cells (Figure 5). We conclude that A77 1726-induced autophagy is mediated by inhibition of S6K1 activity. During preparation of this manuscript, Chen et al. [45] reported that leflunomide induces autophagy in renal cell carcinoma cell lines. Though A77 1726 inhibits PDGF receptor and Src family tyrosine kinases [26, 27], it has no effect on insulin receptor and IGF-1 receptor but rather stimulates IGF-1/IR-induced PI-3 kinase pathway through S6K1-mediated feedback activation [32]. These observations suggest that A77 1726-induced autophagy is not mediated through IGF-1/insulin receptor or other receptor tyrosine kinases, but rather through inhibition of S6K1 activity. In support of this notion, Blommaart et al. [46] showed that phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. Shin et al. [47] reported that silencing S6K1 with siRNA induces autophagy in HEK-293T cells. Consistently, Park et al. [48] reported that PF-4708671 induces autophagy in mouse embryonic fibroblast cells.We further analyzed the signaling pathway of S6K1 suppression-induced autophagy. Kim et al. [40] recently showed that in the TLR-signaling pathway, S6K1 negatively regulates the activity of TAK1. Inokuchi-Shimizu et al. [23] reported that TAK1 deficiency leads to the inhibition of starvation-induced AMPK and ULK1 phosphorylation and activation, subsequently suppressing autophagy in the liver of TAK1-deficientmice. These investigators further showed that TAK1 deficiency also compromises rapamycin-induced autophagy in the hepatocytes of TAK1-knockout mice, indicating that TAK1 is partially required for rapamycin-induced autophagy (Figure 12). Consistent with these observations, we found that inhibition of TAK1 activity by 5Z-7-oxozeaenol or by TAK1 siRNA abrogated A77 1726-induced activation of AMPK and JNK, reduced A77 1726-induced LC3 lipidation. These observations collectively suggest that A77 1726 induces autophagy by inhibiting S6K activity, leading to TAK1 activation, which then activates AMPK and JNK (Figure 12). Of note, S6K1 deficiency leads to AMPK activation in the skeletal muscle tissues and myotubes of S6K1-deficientmice due to increased AMP levels and AMP/ATP ratio [9, 11]. It is not clear if A77 1726-induced AMPK activation is also mediated in part by increased AMP levels and AMP/ATP ratios.
Figure 12
Mechanisms of A77 1726-induced autophagy
A77 1726 inhibits S6K1 activity, leading to TAK1 activation that subsequently activating AMPK and JNK. JNK activation leads to increased p62 expression and disrupts Beclin1 and Bcl2 interaction by phosphorylating Bcl2. Although feedback activation of mTOR by A77 1726 leads to ULK1 phosphorylation at S757, it does not dampen ULK1 phosphorylation at S555 and activation by AMPK. AMPK appears to be at the high end of hormone and metabolic signal pathways that modulates nutrient and energy homeostasis through autophagy.
Mechanisms of A77 1726-induced autophagy
A77 1726 inhibits S6K1 activity, leading to TAK1 activation that subsequently activating AMPK and JNK. JNK activation leads to increased p62 expression and disrupts Beclin1 and Bcl2 interaction by phosphorylating Bcl2. Although feedback activation of mTOR by A77 1726 leads to ULK1 phosphorylation at S757, it does not dampen ULK1 phosphorylation at S555 and activation by AMPK. AMPK appears to be at the high end of hormone and metabolic signal pathways that modulates nutrient and energy homeostasis through autophagy.ULK1 phosphorylation at S757 by mTOR suppresses its activity and autophagy [6, 7]. In contrast, ULK1 phosphorylation at S555 by AMPK leads to ULK1 activation and autophagy. Several other sites including S317, S777, S638, S467, S556, T575 can also be phosphorylated by AMPK but seem to have only marginal effects on the activity of ULK1 [3, 6]. Since inhibition of S6K1 activity by A77 1726 leads to the feedback activation of the PI-3 and MAP kinase pathways through the IGF-1 receptor [32], feedback activation of mTOR by A77 1726 should suppress autophagy. Surprisingly, though ULK1 was highly phosphorylated at S757 due to mTOR feedback activation, A77 1726 did not inhibit but rather induced autophagy. Moreover, A77 1726 induced AMPK phosphorylation at T172 and ULK1 phosphorylation at S555. These observations suggest that AMPK activation through inhibition of S6K1 activity in A77 1726-treated cells was able to blunt the inhibitory effect of feedback-activated mTOR on ULK1 activity. Our further study showed that rapamycin blocked A77 1726-induced ULK1 S757 phosphorylation but did not further increase autophagy (Figure 3). It appears that, when ULK1 is phosphorylated by AMPK at S555 and possibly other sites [3, 49], ULK1 activity can no longer be restrained by S757 phosphorylation. Consistent with our observations, Shang et al. [50] reported that ULK1 S757 phosphorylation transiently regulates autophagy and does not alter the maximum capacity of autophagy after a prolonged starvation. Kang et al. [7] reported that rapamycin is not very effective at inhibiting ULK1 S757 phosphorylation in vitro. Although ULK1 S757 phosphorylation disrupts its interaction with AMPK, we found that mTOR feedback activation and ULK1 S757 phosphorylation did not ablate the interaction between ULK1 and AMPK in A77 1726-treated A375 cells (Figure 5E).Our present study showed that A77 1726 induced JNK phosphorylation and activation in A375 cells in a time- and dose-dependent manner (Figure 9). SP600125 blocked A77 1726-induced Jun phosphorylation and p62 expression. Consistently, S6K1 knockdown and PF-4708671 also led to increased p62 levels as well as JNK and Jun phosphorylation. These results collectively suggest that induction of p62 expression by A77 1726 is mediated by inhibition of S6K1 activity and subsequent activation of JNK. Puissant et al. [38] reported that resveratrol inhibits the PI-3 kinase pathway and induces autophagy in a K562chronic myelogenous leukemia cell line. Interestingly, resveratrol also activates JNK and transcriptionally induces p62 expression [38]. JNK phosphorylates Bcl-2 and promotes its dissociation from Beclin, subsequently promoting autophagy [51] (Figure 12).Our study suggests that increased p62 levels by A77 1726 are due to JNK activation. Interestingly, p62 levels were not decreased in rapamycin-treated A375 cells in which JNK activation was not observed. Although in most cases, induction of autophagy by rapamycin leads to the degradation and decreased levels of p62, Ju et al. [52] reported that rapamycin induces autophagy but does not reduce p62 levels in vitro in C2C12 myotubes and in vivo in the muscle of rapamycin-treated mice. Kim et al. [53] reported that activation of the MAP kinase pathway can up-regulate p62 transcription. Rapamycin induces the feedback activation of the MAP kinase pathway [54], it is likely that rapamycin increases p62 levels through MAP kinase pathway-induced transcriptional regulation of p62. A77 1726 not only induces the feedback activation of the MAP kinase pathway [32] but also strongly activated JNK (Figure 10), and increased p62 expression much stronger than rapamycin. It appears that JNK activation plays a dominant role in mediating A77 1726-induced p62 expression. However, it remains enigmatic why indirect inhibition of S6K1 activity by rapamycin did not activate JNK.He et al. [55] reported that AMPK phosphorylates JNK in vitro, and that AMPK activation by metformin stimulates its interaction with JNK. However, this supposition is challenged since the amino acid sequences in the activation loop sites of JNK do not contain the AMPK consensus, and AMPK does not have a tyrosine kinase activity [44]. Our study showed that the AMPK inhibitor CC failed to block A77 1726-induced JNK activation, while oligomycin and metformin activated AMPK but had no or little effect on JNK activation (Figure 10), suggesting that AMPK is not responsible for activating JNK. On a cautionary note, since CC is not a very specific inhibitor of AMPK [56], the concentration of the compound C we used was relatively low and appeared not capable of inhibiting the major enzyme involved in autophagy. Furthermore, we found that 5Z-7-oxozeaenol and TAK1 siRNA inhibited A77 1726-induced JNK and Jun phosphorylation, suggesting that A77 1726-induced JNK activation is mediated by TAK1 activation (Figure 12). Consistent with this notion, TAK1 plays a critical role in activating the hematopoietic progenitor kinase-1 (HPK1)-induced JNK activation [17].In summary, our study showed that inhibition of S6K1 by A77 1726 led to the activation of AMPK and JNK, both of which contributed to A77 1726-induced autophagy. We further showed that AMPK phosphorylation at T172 and activation led to ULK1 phosphorylation at S555, which overcame the inhibitory effect of ULK1 S757 phosphorylation mediated by feedback-activated mTOR. We further showed that A77 1726 activated AMPK and JNK through S6K1 inhibition-mediated TAK1 activation (Figure 12). Our studies reveal a novel mechanism by which the mTOR-S6K1 pathway connects to the AMPK-ULK1 pathway through TAK1 to regulate autophagy (Figure 12).
MATERIALS AND METHODS
Reagents
Leflunomide and A77 1726 were kindly provided by Cinkate Corporation (Oak Park, IL). Brequinar sodium (BQR), a potent inhibitor of DHO-DHase, was kindly provided by Dupont Corporate (Wilmington, DE). PLX4720 was purchased from Selleck Chemicals Inc. (Houston, TX). SP600125, U0126, PD98059, and LY294002 were 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 and PPP were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies against p62, LC3, ULK1, AMPK, JNK, Jun, AKT, S6K1, S6 and their corresponding phospho-antibodies including ULK1S555, ULK1S757, AMPKT172, AKTS473, S6K1T389, S6S235/236, TAK1T184/187 were purchased from Cell Signaling Technology (Danvers, MA). The expression vector encoding RFP-LC3 (pmRFP-LC3) was purchased from OriGene Technologies, Inc. (Rockville, MD).
Cell lines
A375 is a melanoma cell line with BRAFV600E mutation, wild-type PTEN/PI3KC and p53. MCF-7 is an estrogen receptor-positive breast cancer cell line with PI3KC mutation but with wild-type p53. A375 cells were grown in complete DMEM medium supplemented with 10% fetal bovine serum, streptomycin and penicillin, and L-glutamine. MCF-7 cells were grown in the complete MEM medium supplemented with 10% fetal bovine serum, streptomycin and penicillin, and L-glutamine, non-essential amino acids, and HEPES buffer. C2C12 cells were grown in DMEM supplemented with 10% fetal bovine serum, streptomycin and penicillin, and L-glutamine. For induction of differentiation, the cells were cultured in the complete DMEM medium containing 10% horse serum for 7-10 days. The medium was replenished every three days. All three cell lines were purchased from American Type Culture Collection (Manassas, VA).
Western blot
Cells grown in 6-well plates were harvested 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. After electrophoresis and transfer to Immobolin or nitrocellulose membranes, proteins of interest were probed with their specific antibodies, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG and SuperSignal Western Pico enhanced chemiluminescence substrate (Pierce Chemical Co., Rockford, IL).
S6K1 and TAK1 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 S6K1 expression [57, 58]. TAK1 siRNA was purchased from Cell Signaling Technology (Danvers, MA). A scrambled control siRNA was purchased from Life Technologies (Invitrogen Life Technologies, Grand Island, NY). A375 cells seeded in a 6-well plate were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies, Grand Island, NY) according to the manufacturer’s instruction. After incubation for 48 hr, the cells were harvested and analyzed for S6K1 expression and for the phosphorylation of S6K1, AKT, S6, AMPK, and ULK1 by Western blot.
RFP-LC3 fluorescence analysis
A375 and MCF-7 cells seeded on coverslips were transiently transfected with RFP-LC3 expression plasmid DNA using FuGENE6 following the manufacturer’s protocol. After incubation for 48 hr, the cells were incubated in the presence of A77 1726 (200 μM), rapamycin (20 nM), leflunomide (200 μM) or PF-4708671 (5 μM). After incubation for 16 hr, the cells were fixed in 100% methanol at -20°C for 10 min. The coverslips were mounted with 50% glycerin in PBS containing 4,6-diamidino-2-phenylindole (0.5 μg/ml; Sigma Chemical Co.). Autophagosomes were examined under a Leica LP8 confocal microscope. The autophagosome puncta were examined under a Nikon fluorescence microscope. To determine the effect of S6K1 knockdown on autophagosome formation, A375 cells were transfected with control or S6K1 siRNA as described above. After incubation for 24 hr, the cells were transfected with RFP-LC3 plasmid DNA again. After incubation for another 48 hr, the coverslips were collected, fixed, and mounted on slides and examined for RFP fluorescence under a fluorescent microscope. Autophagosome puncta in A375 cells treated with various drugs or siRNA transfection were counted in 30 randomly selected fields under a 40X objective in a blinded fashion. Results represent the mean ± SD (standard deviation) from three experiments.
Immunoprecipitation
A375 cell lysates in NP-40 lysis buffer were incubated at 4°C with the indicated antibody (2 μg/sample) overnight followed by incubation for 2 hr with Protein A/G-conjugated agarose beads that had been blocked with 5% BSA and washed with NP-40 lysis buffter. The agarose beads were washed 3 times with NP-40 lysis buffer. Immunoprecipitates were analyzed by Western blot with specific antibodies.
Statistical analysis
The differences in the number of puncta in A375 cells treated with various drugs were statistically analyzed by using an unpaired Student t test. A p value of <0.05 was considered statistically significant. All statistics was performed with SigmaPlot 11 software (Systat Software, Inc, San Jose, CA).
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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; 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