Wei Liu1, Yuanyuan Jiang1, Jing Sun1, Shizhong Geng2,3, Zhiming Pan2,3, Richard A Prinz4, Chengming Wang5, Jun Sun6, Xinan Jiao2,3, Xiulong Xu7,8,9. 1. Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, 225009, P. R. China. 2. Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou, 225009, China. 3. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu Province, 225009, China. 4. Department of Surgery, NorthShore University Health System, Evanston, IL, 60201, USA. 5. Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL, 36849, USA. 6. Department of Medicine, University of Illinois at Chicago, Chicago, IL, 60612, USA. 7. Institute of Comparative Medicine, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu Province, 225009, P. R. China. xxl@yzu.edu.cn. 8. Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, Jiangsu Province, 225009, China. xxl@yzu.edu.cn. 9. Department of Cell and Molecular Medicine, Rush University Medical Center, Chicago, IL, 60612, USA. xxl@yzu.edu.cn.
Abstract
Autophagy is a conserved cellular process that functions as a first-line defense to restrict the growth of invading parasitic bacteria. As an intracellular pathogen, Salmonella (S) Typhimurium invades host cells through two Type III secretion systems (T3SS) and resides in the Salmonella-containing vacuole (SCV). When the SCV membrane is perforated and ruptured by T3SS-1, a small portion of the Salmonella egresses from the SCV and replicates rapidly in the nutrient-rich cytosol. Cytosolic Salmonella and those residing in the membrane-damaged SCV are tagged by ubiquitination and marked for autophagy through the ubiquitin-binding adaptor proteins such as p62, NDP52, and optineurin. Prior studies suggest that transient intracellular amino-acid starvation and subsequent inactivation of the mechanistic target of rapamycin (mTOR), a key molecule that phosphorylates Unc-51 like autophagy activating kinase (ULK1) and inhibits its activity, can trigger autophagy in S. Typhimurium-infected cells. Other studies suggest that energy stress in S. Typhimurium-infected cells leads to AMP-activated protein kinase (AMPK) activation and autophagy. In the present study, we report that autophagy was rapidly induced in S. Typhimurium-infected cells, as evidenced by increased LC3 lipidation and decreased p62 levels. However, S. Typhimurium infection drastically increased AKT phosphorylation but decreased S6K1T389, 4E-BPT37/46, and ULK1S757 phosphorylation, suggesting that mTOR activation by AKT is subverted. Further studies showed that AMPK was activated in S. Typhimurium-infected cells, as evidenced by increased ULK1S317 and ACCS79 phosphorylation. AMPK activation was mediated by Toll-like receptor-activated TAK1. Functional studies revealed that AMPK and TAK1 inhibitors accelerated S. Typhimurium growth in HeLa cells. Our results strongly suggest that TAK1 activation leads to AMPK activation, which activates ULK1 by phosphorylating ULK1S317 and suppressing mTOR activity and ULK1S757 phosphorylation. Our study has unveiled a previously unrecognized pathway for S. Typhimurium-induced autophagy.
Autophagy is a conserved cellular process that functions as a first-line defense to restrict the growth of invading parasitic bacteria. As an intracellular pathogen, Salmonella (S) Typhimurium invades host cells through two Type III secretion systems (T3SS) and resides in the Salmonella-containing vacuole (SCV). When the SCV membrane is perforated and ruptured by T3SS-1, a small portion of the Salmonella egresses from the SCV and replicates rapidly in the nutrient-rich cytosol. Cytosolic Salmonella and those residing in the membrane-damaged SCV are tagged by ubiquitination and marked for autophagy through the ubiquitin-binding adaptor proteins such as p62, NDP52, and optineurin. Prior studies suggest that transient intracellular amino-acid starvation and subsequent inactivation of the mechanistic target of rapamycin (mTOR), a key molecule that phosphorylates Unc-51 like autophagy activating kinase (ULK1) and inhibits its activity, can trigger autophagy in S. Typhimurium-infected cells. Other studies suggest that energy stress in S. Typhimurium-infected cells leads to AMP-activated protein kinase (AMPK) activation and autophagy. In the present study, we report that autophagy was rapidly induced in S. Typhimurium-infected cells, as evidenced by increased LC3 lipidation and decreased p62 levels. However, S. Typhimuriuminfection drastically increased AKT phosphorylation but decreased S6K1T389, 4E-BPT37/46, and ULK1S757 phosphorylation, suggesting that mTOR activation by AKT is subverted. Further studies showed that AMPK was activated in S. Typhimurium-infected cells, as evidenced by increased ULK1S317 and ACCS79 phosphorylation. AMPK activation was mediated by Toll-like receptor-activated TAK1. Functional studies revealed that AMPK and TAK1 inhibitors accelerated S. Typhimurium growth in HeLa cells. Our results strongly suggest that TAK1 activation leads to AMPK activation, which activates ULK1 by phosphorylating ULK1S317 and suppressing mTOR activity and ULK1S757 phosphorylation. Our study has unveiled a previously unrecognized pathway for S. Typhimurium-induced autophagy.
Autophagy is a highly conserved self-digestion process that plays a crucial role in maintaining cellular homeostasis in response to nutrient depletion or other cellular stresses such as accumulation of damaged organelles, unneeded protein aggregates, and invading microbes[1-4]. Autophagy is controlled by mTOR and AMP-activated protein kinase (AMPK), two nutrient- and energy-sensitive kinases[5]. These two kinases phosphorylate ULK1/2 at different serine residues and have the opposite effect on ULK activity: mTOR phosphorylates ULK1 at serine 757 (ULK1S757) and inhibits its activity[5-7], whereas AMPK phosphorylates ULK1 at multiple sites, including the serine residues 317, 555, and 777, and activates its activity[6-10]. ULK1/2 binds ATG13 and FIP200 proteins to form a preinitiation complex, which controls the activation of the initiation complex that comprises Beclin 1, ATG14L, VPS34, and VSP15[11-13]. VPS34 is a Class III PI-3 kinase and catalyzes phosphatidylinositol (PI)-4,5 to PI-3-phosphate, which initiates the elongation and nucleation of the double membrane to form autophagosomes[4,14,15].TAK1 is a member of the mitogen-activated protein kinase kinase kinase family and can be activated by multiple extracellular stimuli such as TGF-β, IL-1, tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS)[5]. In addition, microbial proteins and the components of host cell signaling pathways can also regulate TAK1 activity[16]. TAK1 phosphorylates and activates several intracellular kinases, including p38, JNK, and I-kappa B kinase complex (IKK). TAK1 plays important roles in cell survival, differentiation, apoptosis, and inflammatory responses[16]. Emerging evidence suggests that TAK1 activation can induce autophagy in an AMPK-dependent manner[17-20]. Whether TAK1 activation by bacterial LPS is responsible for pathogen-induced AMPK activation and autophagy remains to be defined.Salmonella spp. is a facultative intracellular Gram-negative enteropathogen that causes gastroenteritis and typhoid-like fever[21]. Salmonella enterica serovar Typhimurium (S. Typhimurium) is one of the most common serotypes in human cases of salmonellosis worldwide, despite ongoing implementation of targeted control and prevention measures[22,23]. S. Typhimurium invades intestinal epithelial cells and resides in a specialized niche, the Salmonella-containing vacuole (SCV)[24]. Damage of the SCV membrane by T3SS-1 enables ~25% S. Typhimurium to escape into the cytosol where it can replicate quickly in the nutrient-rich environment. Cytosolic S. Typhimurium is ubiquinated to form a dense ubiquitin chain layer on the surface of Salmonella that peaks 4 h post invasion[25]. Ubiquitin-decorated S. Typhimurium is recognized by multiple autophagy receptors, including NDP52, OPTN, and p62, which bind the LC3-enriched autophore membrane to form autophagosome[26,27]. Recent studies have shown that transient amino-acid starvation due to the cellular membrane damage after Salmonella invasion leads to transient AMPK activation and mTOR inactivation, thus triggering the initiation of autophagy[26,28]. Our present study provides evidence that TAK1 activation is responsible for S. Typhimurium-induced AMPK activation and autophagy. In addition, we found that TAK1-mediated AMPK activation can subvert the AKT-mediated mTOR activation. Thus, our study provides novel insights how S. Typhimurium induces autophagy.
Results
Autophagy induction by S. Typhimurium in HeLa cells
We first verified the ability of S. Typhimurium to induce autophagy in HeLa cells by western blot analysis of LC3 lipidation and p62 degradation. As shown in Fig. 1a,b, S. Typhimurium increased LC3-II lipidation and decreased p62 expression in HeLa cells in a dose- (Fig. 1a) and time- (Fig. 1b) dependent manner. Increased LC3-II lipidation was not due to the stall of autophagy flux since combination of S. Typhimurium with bafilomycin (5 or 20 nM; Fig. 1c) or chloroquine (CQ; 5 or 20 μM; Fig. 1d) increased the levels of LC3-II and the ratios of LC3-II to LC-I, compared to bafilomycin or CQ alone. Bafilomycin (20 nM; Fig. 1c) or CQ (20 μM; Fig. 1d) partially reversed p62 degradation in S. Typhimurium-infected HeLa cells. To further verify the ability of S. Typhimurium to induce autophagy, we analyzed the formation of autophagosomes in RFP-GFP-LC3-transfected HeLa cells. As shown in Fig. 2a, there were very few puncta in the uninfected control cells. In contrast, many autophagosomes accumulated in one side of the cytoplasm close to nuclear membrane in S. Typhimurium-infected cells. There are average 22 puncta per cell in S. Typhimurium-infected HeLa cells (Fig. 2b). Among them, ~70% of the puncta were presented as the red RFP fluorescence dots (Fig. 2b), suggesting the formation of autolysosomes in which the green GFP fluorescence was quenched under the acidic environments. The number of yellow puncta were also significantly higher in bafilomycin- or CQ-treated cells than in untreated control cells. Among them, the majority of puncta in bafilomycin- or CQ-treated HeLa cells were yellow (Fig. 2b), which means that the red and green GFP fluorescence was merged. Both bafilomycin and CQ significantly increased the number of yellow puncta but decreased the number and percent of red puncta in S. Typhimurium-infected cells, suggesting that bafilomycin and CQ block autophagic flux in S. Typhimurium-infected cells.
Fig. 1
Autophagy induction by S. Typhimurium.
HeLa cells were infected with indicated MOI of S. Typhimurium for 2 h (a) or with 10 MOI of S. Typhimurium for the indicated lengths of time (b). Cell lysates were prepared and analyzed for LC3-II lipidation, p62 and actin by western blot with the indicated antibodies. c, d Increased LC3-II lipidation was not due to the stall of autophagy flux. HeLa cells pretreated with bafilomycin (Baf; 5 or 20 nM) or chloroquine (CQ; 5 or 20 μM) for 30 min were infected with S. Typhimurium (10 MOI) for 2 h. Cell lysates were prepared and analyzed for LC3-II lipidation, p62, and actin by western blot with the indicated antibodies. Blots for LC3-II lipidation, p62, and actin were semi-quantified using NIH Image-J software and presented as bar graphs. Data are presented as the mean ± SD (n = 3) relative to control. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection
Fig. 2
S. Typhimurium induces the formation of autolysosomes.
HeLa cells stably expressing the GFP-RFP-LC3 gene were infected with S. Typhimurium (10 MOI) for 2 h in the absence or presence of bafilomycin (20 nM) or CQ (20 μM). The cells were then fixed and stained with DAPI. Autophagosomes presented as the orange puncta and autolysosomes presented as the red puncta were visualized under a confocal microscope (a) and were statistically analyzed (b). Bar length: 20 μm. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection
Autophagy induction by S. Typhimurium.
HeLa cells were infected with indicated MOI of S. Typhimurium for 2 h (a) or with 10 MOI of S. Typhimurium for the indicated lengths of time (b). Cell lysates were prepared and analyzed for LC3-II lipidation, p62 and actin by western blot with the indicated antibodies. c, d Increased LC3-II lipidation was not due to the stall of autophagy flux. HeLa cells pretreated with bafilomycin (Baf; 5 or 20 nM) or chloroquine (CQ; 5 or 20 μM) for 30 min were infected with S. Typhimurium (10 MOI) for 2 h. Cell lysates were prepared and analyzed for LC3-II lipidation, p62, and actin by western blot with the indicated antibodies. Blots for LC3-II lipidation, p62, and actin were semi-quantified using NIH Image-J software and presented as bar graphs. Data are presented as the mean ± SD (n = 3) relative to control. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimuriuminfection
S. Typhimurium induces the formation of autolysosomes.
HeLa cells stably expressing the GFP-RFP-LC3 gene were infected with S. Typhimurium (10 MOI) for 2 h in the absence or presence of bafilomycin (20 nM) or CQ (20 μM). The cells were then fixed and stained with DAPI. Autophagosomes presented as the orange puncta and autolysosomes presented as the red puncta were visualized under a confocal microscope (a) and were statistically analyzed (b). Bar length: 20 μm. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimuriuminfection
Role of mTOR in S. Typhimurium-induced autophagy
Previous studies have established that the sopB protein of S. Typhimurium activates AKT[29-32]. mTOR, a downstream effector activated by AKT, suppresses autophagy by phosphorylating ULK1S757 4,33. We first analyzed the status of AKT phosphorylation as well as several mTOR substrates, including 4E-BPT37/46, S6K1T389, and ULKS757. S. Typhimurium markedly increased AKTS473 phosphorylation in a dose- (Fig. 3a & b) and time-dependent (Fig. 3c & d) manner. Unexpectedly, S. Typhimurium did not correspondingly increase but rather modestly or weakly decreased the phosphorylation of mTORS2448, 4E-BPT37/46, S6S235/236, S6K1T389, and ULKS757 in a dose- (Fig. 3a & b) and time-dependent (Fig. 3c & d) manner. These observations suggest that S. Typhimurium can subvert AKT-mediated mTOR activation, and that blocking of mTOR activity may contribute to Salmonella-induced autophagy.
Fig. 3
Effect of S. Typhimurium on the PI-3 kinase pathway.
HeLa cells were infected with the indicated amount of S. Typhimurium for 2 h (a) or infected with 2 MOI of S. Typhimurium for the indicated length of time (c). Cell lysates were analyzed for AKT, mTOR, ULK1S757, 4EBP1, S6K1, and S6 by western blot with the indicated antibodies. Relative phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software (b and d) and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control
Effect of S. Typhimurium on the PI-3 kinase pathway.
HeLa cells were infected with the indicated amount of S. Typhimurium for 2 h (a) or infected with 2 MOI of S. Typhimurium for the indicated length of time (c). Cell lysates were analyzed for AKT, mTOR, ULK1S757, 4EBP1, S6K1, and S6 by western blot with the indicated antibodies. Relative phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software (b and d) and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control
The TAK1–AMPK pathway is activated in S. Typhimurium-infected cells
It is well established that activation of the Toll-like receptor 4 (TLR4) by LPS leads to TAK1 activation[5]. Emerging evidence suggests that AMPK can be activated by TAK1[17-20]. Here we tested whether S. Typhimuriuminfection could activate TAK1, leading to AMPK activation and autophagy. As shown in Fig. 4, S. Typhimuriuminfection indeed induced TAK1T184/187 autophosphorylation in a dose- (Fig. 4a) and time-dependent (Fig. 4b) manner. TAK1 activation led to increased phosphorylation of its two downstream substrates, AMPKT172 and IKKαS176/180. AMPK phosphorylates multiple serine residues in ULK1[4,14,33]. Interestingly, AMPK activation in Salmonella-infected cells increased ULK1S317 phosphorylation but decreased ULK1S555 phosphorylation in a dose- (Fig. 4a) and time-dependent (Fig. 4b) manner. AMPK activation also led to increased phosphorylation of ACCS79, a well-known substrate of AMPK (Fig. 4).
Fig. 4
Effect of S. Typhimurium on the TAK1–AMPK pathway.
HeLa cells were infected with the indicated amount of MOI of S. Typhimurium for 2 h (a) or were infected with 10 MOI and then incubated for the indicated length of time (b). Cell lysates were analyzed for TAK1, IKK, AMPK, ULK1S555, ULK1S317, and ACCS79 by western blot with the indicated antibodies. Relative phosphorylation levels were semi-quantified using NIH Image-J software and presented as bar graphs. Data are presented as the mean ± SD (n = 3) relative to control. *p < 0.05, **p < 0.01, compared to uninfected control
Effect of S. Typhimurium on the TAK1–AMPK pathway.
HeLa cells were infected with the indicated amount of MOI of S. Typhimurium for 2 h (a) or were infected with 10 MOI and then incubated for the indicated length of time (b). Cell lysates were analyzed for TAK1, IKK, AMPK, ULK1S555, ULK1S317, and ACCS79 by western blot with the indicated antibodies. Relative phosphorylation levels were semi-quantified using NIH Image-J software and presented as bar graphs. Data are presented as the mean ± SD (n = 3) relative to control. *p < 0.05, **p < 0.01, compared to uninfected controlWe next determined whether AMPK was indeed responsible for S. Typhimurium-induced autophagy and the change of ULK1 phosphorylation. As shown in Fig. 5a, compound C (CC), a specific inhibitor of AMPK, blocked S. Typhimurium-induced LC3-II lipidation and p62 degradation. Meanwhile, CC blocked S. Typhimurium-induced AMPKT172, ULK1S317, and ACCS79 phosphorylation and reversed the decrease of ULK1S555 phosphorylation (Fig. 5a). Consistently, CC blocked the formation of autophagosomes and autolysosomes in S. Typhimurium-infected cells (Fig. 5b), as the number of red and yellow fluorescent puncta was significantly lower in S. Typhimurium-infected cells in the presence of CC than in the absence of CC. These observations collectively suggest that AMPK activation plays an important role in S. Typhimurium-induced autophagy.
Fig. 5
Effect of AMPK inhibitor on S. Typhimurium-induced autophagy.
HeLa cells pretreated with CC (1 μM) for 30 min were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the levels of p62 and LC3 and for the phosphorylation of AMPKT172, ULK1S555, ULK1S317, and ACCS79 (a) by western blot with the indicated antibodies or the antibodies against total proteins after stripping. Relative protein and phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection. b, c CC inhibits S. Typhimurium-induced formation of autolysosomes. HeLa cells stably expressing the GFP-RFP-LC3 gene were left uninfected or infected with S. Typhimurium (10 MOI) for 2 h in the absence or presence of CC (1 μM). The cells were then fixed and stained with DAPI. Autophagosomes represented by the orange puncta and autolysosomes represented by the red puncta were visualized under a confocal microscope (b) and statistically analyzed (c). Bar length: 20 μm. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection
Effect of AMPK inhibitor on S. Typhimurium-induced autophagy.
HeLa cells pretreated with CC (1 μM) for 30 min were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the levels of p62 and LC3 and for the phosphorylation of AMPKT172, ULK1S555, ULK1S317, and ACCS79 (a) by western blot with the indicated antibodies or the antibodies against total proteins after stripping. Relative protein and phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimuriuminfection. b, c CC inhibits S. Typhimurium-induced formation of autolysosomes. HeLa cells stably expressing the GFP-RFP-LC3 gene were left uninfected or infected with S. Typhimurium (10 MOI) for 2 h in the absence or presence of CC (1 μM). The cells were then fixed and stained with DAPI. Autophagosomes represented by the orange puncta and autolysosomes represented by the red puncta were visualized under a confocal microscope (b) and statistically analyzed (c). Bar length: 20 μm. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimuriuminfection
Role of TAK1 in AMPK-mediated regulation of autophagy
We then tested whether TAK1 was responsible for S. Typhimurium-induced autophagy and AMPK activation. We first tested whether inhibition of TAK1 activity by 5Z-7-oxozeaenol (5Z), an inhibitor of TAK1, was able to block S. Typhimurium-induced autophagy and AMPK activation. As shown in Fig. 6a, 5Z blocked S. Typhimurium-induced LC3-II lipidation and p62 degradation, blocked S. Typhimurium-induced AMPKT172, ULK1S317, and ACCS79 phosphorylation, and blocked the decrease of ULK1S555 phosphorylation (Fig. 6a). Consistently, 5Z blocked the formation of autophagosomes and autolysosomes in S. Typhimurium-infected cells (Fig. 6b), as the number of red and orange fluorescent puncta was significantly lower in S. Typhimurium-infected cells in the presence of 5Z than in the absence of 5Z (Fig. 6c).
Fig. 6
The effect of TAK1 inhibitor on S. Typhimurium-induced autophagy.
HeLa cells pretreated with 5Z-7-oxozeaenol (5Z; 0.5 μM) for 30 min were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the levels of p62 and LC3 and for the phosphorylation of AMPKT172, ULK1S555, ULK1S317, and ACCS79 (a) by western blot with the indicated antibodies or the antibodies against total proteins after stripping. Relative protein and phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection. b, c 5Z inhibits S. Typhimurium-induced formation of autolysosomes. HeLa cells stably expressing the GFP-RFP-LC3 gene were left uninfected or infected with S. Typhimurium (10 MOI) for 2 h in the absence or presence of 5Z (0.5 μM). The cells were then fixed and stained with DAPI. Autophagosomes represented by the orange puncta and autolysosomes represented by the red puncta were visualized under a confocal microscope (b) and statistically analyzed (c). Bar length: 20 μm. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection
The effect of TAK1 inhibitor on S. Typhimurium-induced autophagy.
HeLa cells pretreated with 5Z-7-oxozeaenol (5Z; 0.5 μM) for 30 min were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the levels of p62 and LC3 and for the phosphorylation of AMPKT172, ULK1S555, ULK1S317, and ACCS79 (a) by western blot with the indicated antibodies or the antibodies against total proteins after stripping. Relative protein and phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimuriuminfection. b, c 5Z inhibits S. Typhimurium-induced formation of autolysosomes. HeLa cells stably expressing the GFP-RFP-LC3 gene were left uninfected or infected with S. Typhimurium (10 MOI) for 2 h in the absence or presence of 5Z (0.5 μM). The cells were then fixed and stained with DAPI. Autophagosomes represented by the orange puncta and autolysosomes represented by the red puncta were visualized under a confocal microscope (b) and statistically analyzed (c). Bar length: 20 μm. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. TyphimuriuminfectionThe role of TAK1 in mediating S. Typhimurium-induced autophagy was further investigated by using TAK1 siRNA. As shown in Fig. 7, TAK1 siRNA effectively suppressed TAK1 expression, and inhibition of TAK1 expression led to the inhibition of S. Typhimurium-induced phosphorylation of TAK1T184/187, AMPKT172, ULK1S317, and ACCS79. TAK1 siRNA blocked S. Typhimurium-induced LC3-II lipidation and p62 degradation, and restored ULK1S555 phosphorylation in S. Typhimurium-infected HeLa cells (Fig. 7). These observations collectively suggest that TAK1 plays a critical role in mediating S. Typhimurium-induced activation of AMPK.
Fig. 7
The effect of TAK1 siRNA on S. Typhimurium-induced autophagy.
HeLa cells were transfected with control or TAK1 siRNA. After incubation for 48 h, the cells were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the levels of p62 and LC3 and for the phosphorylation of AMPKT172, ULK1S555, ULK1S317, and ACCS79 by western blot with the indicated antibodies or the antibodies against total proteins after stripping. Relative protein and phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection
The effect of TAK1 siRNA on S. Typhimurium-induced autophagy.
HeLa cells were transfected with control or TAK1 siRNA. After incubation for 48 h, the cells were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the levels of p62 and LC3 and for the phosphorylation of AMPKT172, ULK1S555, ULK1S317, and ACCS79 by western blot with the indicated antibodies or the antibodies against total proteins after stripping. Relative protein and phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimuriuminfection
AMPK activation circumvents mTOR activity
It has been well documented that AMPK activation can lead to the suppression of mTOR activity by phosphorylating Raptor[4]. Here we tested whether subversion of AKT-mediated mTOR activation in S. Typhimurium-infected HeLa cells was due to TAK1-activated AMPK. As shown in Fig. 8, S. Typhimurium modestly or weakly decreased ULK1S757, S6K1T389, 4E-BPT37/46, and S6S235/236 phosphorylation, which was blocked by CC (Fig. 8a), 5Z (Fig. 8b), or TAK1 siRNA (Fig. 8c). These observations suggest that AKT-mediated mTOR activation is subverted by TAK1-activated AMPK.
Fig. 8
TAK1-medidated AMPK activation circumvents mTOR activity induced by S. Typhimurium.
HeLa cells were pretreated with CC (1 μM) or 5Z (0.5 μM), or transiently transfected with control or TAK1 siRNA for 48 h. The cells were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the phosphorylation of ULK1S757, S6K1, S6, and 4E-BP (a, c) by western blot with the indicated antibodies. Relative phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimurium infection
TAK1-medidated AMPK activation circumvents mTOR activity induced by S. Typhimurium.
HeLa cells were pretreated with CC (1 μM) or 5Z (0.5 μM), or transiently transfected with control or TAK1 siRNA for 48 h. The cells were left uninfected or infected with S. Typhimurium (10 MOI). After incubation for 2 h, the cell lysates were prepared and analyzed for the phosphorylation of ULK1S757, S6K1, S6, and 4E-BP (a, c) by western blot with the indicated antibodies. Relative phosphorylation levels were analyzed by quantification of the density of the protein bands with NIH Image-J software and presented as bar graphs. *p < 0.05, **p < 0.01, compared to uninfected control; #p < 0.05, ##p < 0.01, compared to S. Typhimuriuminfection
The effect of autophagy on bacterial invasion and replication
Finally, we tested whether inhibition of the TAK1–AMPK pathway would lead to increased S. Typhimurium replication. As shown in Fig. 9a, infectionTAK1 inhibitor 5Z and AMPK inhibitor CC significantly increased the number of S. Typhimurium in HeLa cells at 2, 4, and 8 h post infection. However, neither 5Z nor AMPK had any effect on the growth of S. Typhimurium in vitro in LB medium (Fig. 9b). These observations suggest that the inhibitory effect of 5Z and CC on Salmonella replication in HeLa cells is likely mediated by autophagy suppression.
Fig. 9
The effect of TAK1 and AMPK inhibitors on S. Typhimurium growth.
a HeLa cells were left untreated or pretreated with CC (1 μM) or 5Z (0.5 μM) for 30 min. The cells were then infected with S. Typhimurium (10 MOI). After incubation for the indicated lengths of time, the cells were harvested and lysed. The colony formation units were analyzed by counting the number of bacterial colonies grown in the LB plates. The results represent the mean ± SD from the triplicate from one of three experiments with similar results. *p < 0.05, **p < 0.01, compared to uninfected control. b
S. Typhimurium inoculated in LB medium (100 μl) was cultured in the absence or presence of CC (0.5 μM) or 5Z (1 μM) at 37 °C for 8 h with agitation. The OD600 values of triplicate cultures in LB medium were determined in the indicated intervals of indicated time. Gentamycin (100 μg/ml) was used as a positive control. c Schematic mode of Salmonella-induced autophagy. The binding of the TLR4-MD2 complex by LPS, which is abundantly present in the wall of the Gram-negative bacteria such as S. Typhimurium, activates TRAF6 through its two adaptor proteins, MyD88 and TRIF. In addition, the binding of TLR5 and TLR9 by flagelin and CpG, respectively, can also activates TRAF6 through MyD88. As a E3 ubiquitin ligase, TAFF6 induces TAK1 K63-ubiquitination and activation, leading to NF-kB and AMPK activation, the former regulates the expression of inflammatory cytokines, whereas the latter activates ULK1 and regulates autophagy. AKT activation by the sopB protein of S. Typhimurium would activate its downstream effector mTOR and subsequently suppress autophagy. In our model, AMPK activation by TAK1 circumvents the AKT-mediated mTOR activation by phosphorylating Raptor, a subunit in the mTORC1 complex. Inactivation of mTOR activity suppresses ULK1S757 phosphorylation and induces ULK1 activation and autophagy
The effect of TAK1 and AMPK inhibitors on S. Typhimurium growth.
a HeLa cells were left untreated or pretreated with CC (1 μM) or 5Z (0.5 μM) for 30 min. The cells were then infected with S. Typhimurium (10 MOI). After incubation for the indicated lengths of time, the cells were harvested and lysed. The colony formation units were analyzed by counting the number of bacterial colonies grown in the LB plates. The results represent the mean ± SD from the triplicate from one of three experiments with similar results. *p < 0.05, **p < 0.01, compared to uninfected control. b
S. Typhimurium inoculated in LB medium (100 μl) was cultured in the absence or presence of CC (0.5 μM) or 5Z (1 μM) at 37 °C for 8 h with agitation. The OD600 values of triplicate cultures in LB medium were determined in the indicated intervals of indicated time. Gentamycin (100 μg/ml) was used as a positive control. c Schematic mode of Salmonella-induced autophagy. The binding of the TLR4-MD2 complex by LPS, which is abundantly present in the wall of the Gram-negative bacteria such as S. Typhimurium, activates TRAF6 through its two adaptor proteins, MyD88 and TRIF. In addition, the binding of TLR5 and TLR9 by flagelin and CpG, respectively, can also activates TRAF6 through MyD88. As a E3 ubiquitin ligase, TAFF6 induces TAK1 K63-ubiquitination and activation, leading to NF-kB and AMPK activation, the former regulates the expression of inflammatory cytokines, whereas the latter activates ULK1 and regulates autophagy. AKT activation by the sopB protein of S. Typhimurium would activate its downstream effector mTOR and subsequently suppress autophagy. In our model, AMPK activation by TAK1 circumvents the AKT-mediated mTOR activation by phosphorylating Raptor, a subunit in the mTORC1 complex. Inactivation of mTOR activity suppresses ULK1S757 phosphorylation and induces ULK1 activation and autophagy
Discussion
Autophagy cooperates with innate immunity to clear the intracellular bacteria[34,35]. Pattern recognition receptors such as the TLRs can cross-activate the autophagic pathway[36-40]. The underlying molecular mechanisms remain elusive. TAK1 is a serine/threonine kinase activated by numerous inflammatory cytokines and by TLR through the ubiquitin ligase TRAF6 (Fig. 9c)[5]. Although emerging evidence suggests that TAK1 can activate AMPK to induce autophagy[17-20], whether TAK1 activation by bacteria is responsible for autophagy induction has not been investigated. In the present study, we demonstrated that TAK1 activity was required for AMPK activation and autophagy induction in S. Typhimurium-infected HeLa cells. We postulate that the binding of TLR4, TLR5, and TLR9 by LPS, flagellin, and CpG of S. Typhimurium, respectively, activates TRAF6 through Myd88 and/or TRIF, leading to TAK1 and AMPK activation. AMPK induces autophagy by activating ULK1 through phosphorylating ULK1S317 but blocks ULK1S757 phosphorylation by suppressing mTOR1 activity (Fig. 9c). Our study has unveiled a previously unrecognized signaling pathway that plays a central role in Salmonella-induced autophagy.AMPK is a crucial kinase in autophagy regulation. AMPK senses the intracellular energy state[4]. When the ratios of intracellular AMP/ATP levels are increased, AMPK is then activated. In addition, AMPK can be activated by three protein kinases, including LKB1, calcium/calmodulin-dependent kinase kinase, and TAK1[4,14,33]. AMPK activates ULK1 by phosphorylating ULK1 at multiple serine residues, including S317, S555, S777, and S468[4,14,33]. In addition, AMPK can indirectly activate ULK1 by suppressing mTOR activity and decreasing ULK1S757 phosphorylation[41-43]. A recent study by Ganesan et al.[44]. reported that the S. Typhimurium SL1344 transiently activates AMPK activity 1 h post infection in mouse bone marrow-derived macrophages by decreasing the ATP levels. Further studies demonstrated that AMPK along with its upstream effectors, LKB kinase and SIRT1 deacetylase, are re-located into the membrane of autolysosomes where they are degraded by lysosomal proteases. AMPK downregulation thus restrains the sustained AMPK activation and autophagy, as evidenced by increased LC3 lipidation and decreased p62 levels, which only transiently occur ~1 h after S. Typhimuriuminfection[44]. Our present study demonstrated that increased AMPK phosphorylation and LC3-II lipidation as well as decreased p62 levels were maintained in S. Typhimurium-infected HeLa cells for at least 2 h. In addition, AMPK protein levels were not decreased during this time period. It is not clear whether the difference in AMPK activation and autophagy kinetics in S. Typhimurium-infected bone marrow-derived macrophages and HeLa cells is due to the different cell types. Nevertheless, both studies suggest that AMPK activation plays a critical role in S. Typhimurium-induced autophagy.The TLRs have been implicated in regulating autophagy[35,38,40]. For example, activation of TLR4 and TLR3 by LPS and polyinosinic–polycytidylic acid, respectively, induced autophagy in macrophages and lung cancer cells[37,45]. A recent study by McCarthy et al. showed that the TAK1–AMPK pathway is activated by TLR9 in vascular smooth muscle cells[46]. Several earlier studies indicate that TLR4 activation by LPS, which is abundantly present on the cell walls of Gram-negative bacteria, induces autophagy through TRIF-mediated disruption of the Bcl-2-Beclin1 interaction[37,45]. Although it has been long recognized that the availability of Beclin1 alone is not sufficient to trigger autophagy, what other signaling pathways activated by TLR lead to autophagy induction remains largely uncharacterized[35,38,40]. TLR4 activation by LPS activates NF-κB through TAK1-mediated IKK activation (Fig. 9c)[4,5]. In addition to activating IKK, TAK1 can also activate AMPK by phosphorylating T172, which subsequently induces autophagy[4]. For example, Ding et al.[47] reported earlier that TAK1 is required for TGF-β-induced autophagy in murine mesangial cells. Herrero-Martin et al.[18] reported that TNF-related apoptosis-inducing ligand (TRAIL) induces autophagy in human epithelial cells by AMPK in a TAK1-dependent and LKB1-independent manner. Xie et al. reported that AMPK activation is blocked in TAK1-deficientmouse embryos and mouse fibroblasts[20]. TAK1 activates AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells[18]. TAK1 is responsible for VEGF–induced AMPK activation in endothelial cells[48]. We recently reported that TAK1 is also responsible for S6K1 inhibition-induced AMPK activation and autophagy[49]. Our present study showed that TAK1 was activated in Salmonella-infected HeLa cells in a time- and dose-dependent manner, and that TAK1 activation led to increased IKKα and AMPK phosphorylation. We further demonstrated that TAK1 siRNA and 5Z blocked Salmonella-induced AMPK activation and autophagy. These observations collectively suggest that TAK1 plays a critical role in S. Typhimurium-induced AMPK activation and autophagy.The binding of TLR2/1, TLR2/6, TLR4, TLR5, and TLR9 by diacyl lipoproteins, triacyl lipoproteins, LPS, flagelin, and CpG, respectively, can readily activate TAK1[50]. It is noteworthy that TAK1-mediated AMPK activation in S. Typhimurium-infected HeLa cells did not increase ULK1S555 but rather increased ULK1S317 phosphorylation. This observation concurs with a previous study showing that ULK1 is phosphorylated at S317 and S777 but not at S555 in HEK293 cells under glucose starvation[6]. Interestingly, ULK1S555 phosphorylation was increased in Salmonella-infectedRAW264.7 cells, a murine macrophage cell line (Liu et al., unpublished observations). Ganesan et al.[44] reported that S. Typhimuriuminfection of bone marrow-derived macrophages causes energy depletion, as evidenced by decreased intracellular ATP levels. Macrophages are very sensitive to S. Typhimurium killing due to necroptosis and pyroptosis. We did not analyze intracellular AMP and ATP levels in S. Typhimurium-infected HeLa cells. It is not clear whether decreased energy levels may also contribute to AMPK activation in HeLa cells.mTOR plays a critical role in regulating autophagy[51]. mTOR phosphorylates ULK1S757 and inhibits its activity, leading to the suppression of autophagy[4,33]. Absence of ULK1S757 phosphorylation under nutrient depletion leads to ULK1 activation and autophagy induction[4,33]. It is well established that sopB of S. Typhimurium activates AKT in both macrophages and in epithelial tumor cell lines[29-32]. In addition, AKT can be targeted by S. Typhimurium to the SCV membrane of peritoneal exudate macrophages and activated by FAK[52]. Thus, AKT/mTOR activation has been considered a mechanism by which Salmonella evades autophagy-mediated destruction of intracellular bacteria[52]. Tattoli et al.[53] reported that AKT is activated in HeLa cells; it is not clear whether mTOR is correspondingly activated since phosphorylation is decreased in one of its substrates, 4E-BP, but is increased in another substrate, S6K1, as well as S6. Although the status of ULK1S757 is not known, these investigators suggest that transient amino-acid starvation leads to the suppression of mTOR activity[53]. Genesan et al. reported that phosphorylation of AKT and S6K1, a downstream substrate of mTOR, is persistently increased in S. Typhimurium-infected bone marrow-derived macrophages[54]. In the present study, we demonstrated that, although AKT phosphorylation was remarkably increased in S. Typhimurium-infected HeLa cells in a dose- and time-dependent manner, several substrates of mTOR, including S6K1T389, 4E-BPT37/46, and ULK1S757, were not correspondingly increased but rather modestly or weakly decreased in a dose- and time-dependent manner. On the basis of the well-established negative regulation of mTORC1 by AMPK phosphorylation of Raptor[55,56], we propose that blockade of mTOR activation in S. Typhimurium-induced HeLa cells is due to the inhibition of mTORC1 activity by AMPK. Indeed, increased phosphorylation of S6K1T389, ULK1S757, and 4E-BPT37/46 was detected in S. Typhimurium-infected HeLa cells in the presence of either a TAK1 or AMPK inhibitor as well as TAK1 siRNA. These observations suggest that AMPK activation by TAK1 can subdue AKT-mediated mTOR activation, leading to decreased ULK1S757 and S6K1T389 phosphorylation in S. Typhimurium-infected HeLa cells (Fig. 9c).Autophagy plays an important role in restricting S. Typhimurium bacterial growth in epithelial cells and macrophages. S. Typhimurium grows faster in ATG4-deficient murine embryonic fibroblast cells than in the wild-type cells[57]. Optineurin is an important autophagic receptor in S. Typhimurium invasion. The colonization rate of S. Typhimurium is twofold higher in the OPTN-deficient cells[58]. Genetic inactivation of the autophagic pathway by ATG1, ATG6, and ATG7 gene knockout increases intracellular bacterial replication and decreases the lifespan of two model organisms, Caenorhabditis elegans and Dictyostelium disodium[59]. Consistently, there are 100-fold more S. Typhimurium bacteria in the mesangial lymph nodes and spleen of the mice with ATG16L1 conditionally knocked out in the intestinal epithelial cells than in that of wild-type mice[60]. Trifluoperazine, an autophagy activator, inhibits the replication of S. Typhimurium in HeLa cells[60]. S. Typhimurium bacterial burdens are ~100-fold heavier in the spleen and liver of mice, with ATG5 being conditionally knocked out in the intestinal epithelial cells[61]. Our present study showed that AMPK and TAK1 inhibitors, both of which inhibited S. Typhimurium-induced autophagy, accelerated bacterial growth in HeLa cells, suggesting that autophagy can restrict intracellular bacterial growth.We are aware of several weaknesses in our current study. First, while HeLa cells have been widely used as a model system for studying the mechanisms of Salmonella-induced autophagy, the results might be different in the relevant cell types of Salmonella infection such as macrophages and intestinal epithelial cells. Secondly, the role of TAK1 in mediating Salmonella-induced AMPK activation and autophagy and in restricting bacterial growth was not investigated in vivo in TAK1-deficientmice. These in vitro observations need to be verified in vivo in a mouse model. Thirdly, while prior extensive evidence suggests that TAK1 is activated through TLR4, the role of TLR4 as well as its adaptor proteins such as MyD88 and TRIF in mediating Salmonella-activated TAK1–AMPK pathway was not investigated in the present study. Data from these detailed studies should further strengthen our conclusion.In summary, our present study has provided unambiguous evidence that AMPK is activated in S. Typhimurium-infected HeLa cells, and that TAK1, a kinase activated by multiple TLRs such as TLR4 by LPS, TLR5 by flagellin, and TLR9 by CpG, is primarily responsible for AMPK activation (Fig. 9c). Activated AMPK phosphorylates ULK1S317 and meanwhile circumvents AKT-mediated mTOR activation, leading to decreased ULK1S757 phosphorylation. Thus, ULK1 is activated through increased phosphorylation at S317 and decreased phosphorylation at S757. ULK1 activation plays an important role in the autophagy initiation in Salmonella-infected cells. Our study establishes a previously unrecognized link between the TLR signaling and autophagic pathways (Fig. 9c).
Materials and methods
Reagents
Bafilomycin, CQ, and 5Z were purchased from Sigma (St. Louis, MO). CC was purchased from Selleck Inc. (Houston, TX). Anti-actin mAb was purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies against LC3, ULK1, AMPK, mTOR, AKT, S6K1, 4E-BP, S6, ACC (acetyl-CoA carboxylase), TAK1, and their corresponding phospho-antibodies including ULK1S555, ULK1S757, ULK1S317, AMPKT172, mTORS2448, AKTS473, S6K1T389, S6S235/236, 4E-BPT37/46, IKKαS176/180, ACCS79, and TAK1T184/187 were purchased from Cell Signaling Technology (Danvers, MA).
Cells
HeLa cells were purchased from American Tissue Culture Collection (Manassas, VA). The cells were grown in the complete DMEM medium supplemented with 10% fetal bovine serum, streptomycin and penicillin, and L-glutamine. HeLa cells infected with a lentiviral vector encoding the pLV-LC3-GFP-RFP gene (Chengdu Transvector Biotechnology Inc., Chengdu, China) were selected in the complete medium containing puromycin (1.5 μg/ml; Life Technologies).
Bacteria
S. Typhimurium wild-type strain SL1344 was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (NICPBP), China. Bacteria were grown nonagitated in 10 ml of Luria-Bertani (LB) broth with 0.01 ml of a stationary-phase culture, followed by overnight incubation (>18 h) at 37 °C[62].
Infection of cells
HeLa cells seeded in six-well plates were infected with the indicated multiplicity of infection (MOI) of S. Typhimurium (SL1344). After 30 min, extracellular bacteria were removed. The cells were incubated for 30 min in the medium containing 100 μg/ml of gentamicin and then were washed and subsequently cultured in the medium containing gentamycin (10 μg/ml) for the indicated length of time. The cells were harvested and analyzed by western blot with the indicated antibodies.
Western blot
Cells grown in six-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 r.p.m. for 15 min. Cell lysates were analyzed by western blot with antibodies against the proteins of interest, followed by horseradish peroxidase-conjugated goat anti-rabbit IgG and SuperSignal Western Pico enhanced chemiluminoscence substrate (Pierce Chemical Co., Rockford, IL). The density of the bands was analyzed by using NIH Image-J software and normalized by the arbitrary units of their corresponding total proteins or β-actin as indicated. Quantified results were presented as the mean ± SD from three experiments in bar graphs.
TAK1 knockdown
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). HeLa cells seeded in a six-well plate were transfected with siRNA using Lipofectamine RNAiMAX (Invitrogen Life Technologies) according to the manufacturer’s instruction. After incubation for 48 h, the cells were left uninfected or infected with 10 MOI and then incubated for 2 h. The cell lysates were prepared and analyzed for the expression of TAK1 and other relevant proteins.
Bacterial colonization
To determine the effect of CC and 5Z-7-oxozeaenol on S. Typhimurium colonization in LB, S. Typhimurium was prepared as described above. An aliquot of 100 μl bacterial cultures were grown in liquid LB medium, or LB with CC (0.5 μM) /5Z-7-oxozeaenol (1 μM), at 37 °C for 8 h with agitation. The OD600 values of triplicate cultures in LB medium were determined in 1-h intervals. To ascertain bacterial invasion, RAW264.7 and HeLa cells were seeded in 24-well plates, and the cells were infected with S. Typhimurium as described above. Cells were incubated for an additional 120 min in DMEM with gentamicin, washed, and incubated with shaking in HBSS containing Triton X-100 in a cold room. Bacterial CFU were determined by plating diluted cell lysates onto MacConkey agar culture plates (Difco Laboratories Inc.) and incubating the cultures at 37 °C overnight.
Autophagosome analysis
HeLa cells stably transfected with GFP-RFP-LC3 were seeded on coverslips. After infection of S. Typhimurium, the cells were incubated in the absence or presence of CC (1 μM) or 5Z-7-oxozeaenol (0.5 μM). After incubation for 2 h, the cells were fixed in 4% paraformaldehyde at room temperature for 10 min. The coverslips were mounted with 50% glycerin in PBS4,6-diamidino-2-phenylindole (0.5 μg/ml; Sigma Chemical Co.). Autophagosomes were examined under a Leica LP8 confocal microscope. The red and orange puncta in the cells of 10 random fields (100×) were counted in a blinded manner. Results represent the mean puncta per cell ± SD from one of three independent experiments with similar results. Percent red puncta = the number of red puncta ÷ (the number of red puncta + the number of orange puncta) × 100%
Statistical analysis
All statistics was performed with SigmaPlot 11 software (Systat Software Inc, San Jose, CA). The differences in the number of puncta in HeLa cells and the density of scanned bands were statistically analyzed by using an unpaired Student's t-test. A p value of <0.05 was considered statistically significant.
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Susanna Ambrosio; Amal O Amer; Veena Ammanathan; Zhenyi An; Stig U Andersen; Shaida A Andrabi; Magaiver Andrade-Silva; Allen M Andres; Sabrina Angelini; David Ann; Uche C Anozie; Mohammad Y Ansari; Pedro Antas; Adam Antebi; Zuriñe Antón; Tahira Anwar; Lionel Apetoh; Nadezda Apostolova; Toshiyuki Araki; Yasuhiro Araki; Kohei Arasaki; Wagner L Araújo; Jun Araya; Catherine Arden; Maria-Angeles Arévalo; Sandro Arguelles; Esperanza Arias; Jyothi Arikkath; Hirokazu Arimoto; Aileen R Ariosa; Darius Armstrong-James; Laetitia Arnauné-Pelloquin; Angeles Aroca; Daniela S Arroyo; Ivica Arsov; Rubén Artero; Dalia Maria Lucia Asaro; Michael Aschner; Milad Ashrafizadeh; Osnat Ashur-Fabian; Atanas G Atanasov; Alicia K Au; Patrick Auberger; Holger W Auner; Laure Aurelian; Riccardo Autelli; Laura Avagliano; Yenniffer Ávalos; Sanja Aveic; Célia Alexandra Aveleira; Tamar Avin-Wittenberg; Yucel Aydin; Scott Ayton; Srinivas Ayyadevara; Maria Azzopardi; Misuzu Baba; Jonathan M Backer; Steven K Backues; Dong-Hun Bae; 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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; Bo Wang; Chao-Yung Wang; Chen Wang; Chenran Wang; Chenwei Wang; Cun-Yu Wang; Dong Wang; Fangyang Wang; Feng Wang; Fengming Wang; Guansong Wang; Han Wang; Hao Wang; Hexiang Wang; Hong-Gang Wang; Jianrong Wang; Jigang Wang; Jiou Wang; Jundong Wang; Kui Wang; Lianrong Wang; Liming Wang; Maggie Haitian Wang; Meiqing Wang; Nanbu Wang; Pengwei Wang; Peipei Wang; Ping Wang; Ping Wang; Qing Jun Wang; Qing Wang; Qing Kenneth Wang; Qiong A Wang; Wen-Tao Wang; Wuyang Wang; Xinnan Wang; Xuejun Wang; Yan Wang; Yanchang Wang; Yanzhuang Wang; Yen-Yun Wang; Yihua Wang; Yipeng Wang; Yu Wang; Yuqi Wang; Zhe Wang; Zhenyu Wang; Zhouguang Wang; Gary Warnes; Verena Warnsmann; Hirotaka Watada; Eizo Watanabe; Maxinne Watchon; Anna Wawrzyńska; Timothy E Weaver; Grzegorz Wegrzyn; Ann M Wehman; Huafeng Wei; Lei Wei; Taotao Wei; Yongjie Wei; Oliver H Weiergräber; Conrad C Weihl; Günther Weindl; Ralf Weiskirchen; Alan Wells; Runxia H Wen; Xin Wen; Antonia Werner; Beatrice Weykopf; Sally P Wheatley; J Lindsay Whitton; Alexander J Whitworth; Katarzyna Wiktorska; Manon E Wildenberg; Tom Wileman; Simon Wilkinson; Dieter Willbold; Brett Williams; Robin S B Williams; Roger L Williams; Peter R Williamson; Richard A Wilson; Beate Winner; Nathaniel J Winsor; Steven S Witkin; Harald Wodrich; Ute Woehlbier; Thomas Wollert; Esther Wong; Jack Ho Wong; Richard W Wong; Vincent Kam Wai Wong; W Wei-Lynn Wong; An-Guo Wu; Chengbiao Wu; Jian Wu; Junfang Wu; Kenneth K Wu; Min Wu; Shan-Ying Wu; Shengzhou Wu; Shu-Yan Wu; Shufang Wu; William K K Wu; Xiaohong Wu; Xiaoqing Wu; Yao-Wen Wu; Yihua Wu; Ramnik J Xavier; Hongguang Xia; Lixin Xia; Zhengyuan Xia; Ge Xiang; Jin Xiang; Mingliang Xiang; Wei Xiang; Bin Xiao; Guozhi Xiao; Hengyi Xiao; Hong-Tao Xiao; Jian Xiao; Lan Xiao; Shi Xiao; Yin Xiao; Baoming Xie; Chuan-Ming Xie; Min Xie; Yuxiang Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Congfeng Xu; En Xu; Haoxing Xu; Jing Xu; JinRong Xu; Liang Xu; Wen Wen Xu; Xiulong Xu; Yu Xue; Sokhna M S Yakhine-Diop; Masamitsu Yamaguchi; Osamu Yamaguchi; Ai Yamamoto; Shunhei Yamashina; Shengmin Yan; Shian-Jang Yan; Zhen Yan; Yasuo Yanagi; Chuanbin Yang; Dun-Sheng Yang; Huan Yang; Huang-Tian Yang; Hui Yang; Jin-Ming Yang; Jing Yang; Jingyu Yang; Ling Yang; Liu Yang; Ming Yang; Pei-Ming Yang; Qian Yang; Seungwon Yang; Shu Yang; Shun-Fa Yang; Wannian Yang; Wei Yuan Yang; Xiaoyong Yang; Xuesong Yang; Yi Yang; Ying Yang; Honghong Yao; Shenggen Yao; Xiaoqiang Yao; Yong-Gang Yao; Yong-Ming Yao; Takahiro Yasui; Meysam Yazdankhah; Paul M Yen; Cong Yi; Xiao-Ming Yin; Yanhai Yin; Zhangyuan Yin; Ziyi Yin; Meidan Ying; Zheng Ying; Calvin K Yip; Stephanie Pei Tung Yiu; Young H Yoo; Kiyotsugu Yoshida; Saori R Yoshii; Tamotsu Yoshimori; Bahman Yousefi; Boxuan Yu; Haiyang Yu; Jun Yu; Jun Yu; Li Yu; Ming-Lung Yu; Seong-Woon Yu; Victor C Yu; W Haung Yu; Zhengping Yu; Zhou Yu; Junying Yuan; Ling-Qing Yuan; Shilin Yuan; Shyng-Shiou F Yuan; Yanggang Yuan; Zengqiang Yuan; Jianbo Yue; Zhenyu Yue; Jeanho Yun; Raymond L Yung; David N Zacks; Gabriele Zaffagnini; Vanessa O Zambelli; Isabella Zanella; Qun S Zang; Sara Zanivan; Silvia Zappavigna; Pilar Zaragoza; Konstantinos S Zarbalis; Amir Zarebkohan; Amira Zarrouk; Scott O Zeitlin; Jialiu Zeng; Ju-Deng Zeng; Eva Žerovnik; Lixuan Zhan; Bin Zhang; Donna D Zhang; Hanlin Zhang; Hong Zhang; Hong Zhang; Honghe Zhang; Huafeng Zhang; Huaye Zhang; Hui Zhang; Hui-Ling Zhang; Jianbin Zhang; Jianhua Zhang; Jing-Pu Zhang; Kalin Y B Zhang; Leshuai W Zhang; Lin Zhang; Lisheng Zhang; Lu Zhang; Luoying Zhang; Menghuan Zhang; Peng Zhang; Sheng Zhang; Wei Zhang; Xiangnan Zhang; Xiao-Wei Zhang; Xiaolei Zhang; Xiaoyan Zhang; Xin Zhang; Xinxin Zhang; Xu Dong Zhang; Yang Zhang; Yanjin Zhang; Yi Zhang; Ying-Dong Zhang; Yingmei Zhang; Yuan-Yuan Zhang; Yuchen Zhang; Zhe Zhang; Zhengguang Zhang; Zhibing Zhang; Zhihai Zhang; Zhiyong Zhang; Zili Zhang; Haobin Zhao; Lei Zhao; Shuang Zhao; Tongbiao Zhao; Xiao-Fan Zhao; Ying Zhao; Yongchao Zhao; Yongliang Zhao; Yuting Zhao; Guoping Zheng; Kai Zheng; Ling Zheng; Shizhong Zheng; Xi-Long Zheng; Yi Zheng; Zu-Guo Zheng; Boris Zhivotovsky; Qing Zhong; Ao Zhou; Ben Zhou; Cefan Zhou; Gang Zhou; Hao Zhou; Hong Zhou; Hongbo Zhou; Jie Zhou; Jing Zhou; Jing Zhou; Jiyong Zhou; Kailiang Zhou; Rongjia Zhou; Xu-Jie Zhou; Yanshuang Zhou; Yinghong Zhou; Yubin Zhou; Zheng-Yu Zhou; Zhou Zhou; Binglin Zhu; Changlian Zhu; Guo-Qing Zhu; Haining Zhu; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Yanping Zhu; Yushan Zhu; Haixia Zhuang; Xiaohong Zhuang; Katarzyna Zientara-Rytter; Christine M Zimmermann; Elena Ziviani; Teresa Zoladek; Wei-Xing Zong; Dmitry B Zorov; Antonio Zorzano; Weiping Zou; Zhen Zou; Zhengzhi Zou; Steven Zuryn; Werner Zwerschke; Beate Brand-Saberi; X Charlie Dong; Chandra Shekar Kenchappa; Zuguo Li; Yong Lin; Shigeru Oshima; Yueguang Rong; Judith C Sluimer; Christina L Stallings; Chun-Kit Tong Journal: Autophagy Date: 2021-02-08 Impact factor: 13.391
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