Inhibition of the ATR kinase has emerged as a therapeutically attractive means to target cancer since the development of potent inhibitors, which are now in clinical testing. We investigated a potential link between ATR inhibition and the autophagy process in esophageal cancer cells using four ATR inhibitors including two in clinical testing. The response to pharmacological ATR inhibitors was compared with genetic systems to investigate the ATR dependence of the effects observed. The ATR inhibitor, VX-970, was found to lead to an accumulation of p62 and LC3-II indicative of a blocked autophagy. This increase in p62 occurred post-transcriptionally and in all the cell lines tested. However, our data indicate that the accumulation of p62 occurred in an ATR-independent manner and was instead an off-target response to the ATR inhibitor. This study has important implications for the clinical response to pharmacological ATR inhibition, which in some cases includes the blockage of autophagy.
Inhibition of the ATR kinase has emerged as a therapeutically attractive means to target cancer since the development of potent inhibitors, which are now in clinical testing. We investigated a potential link between ATR inhibition and the autophagy process in esophageal cancer cells using four ATR inhibitors including two in clinical testing. The response to pharmacological ATR inhibitors was compared with genetic systems to investigate the ATR dependence of the effects observed. The ATR inhibitor, VX-970, was found to lead to an accumulation of p62 and LC3-II indicative of a blocked autophagy. This increase in p62 occurred post-transcriptionally and in all the cell lines tested. However, our data indicate that the accumulation of p62 occurred in an ATR-independent manner and was instead an off-target response to the ATR inhibitor. This study has important implications for the clinical response to pharmacological ATR inhibition, which in some cases includes the blockage of autophagy.
Ataxia telangiectasia and Rad3-related (ATR) is an essential kinase that plays a pivotal role in the cellular response to replication stress and DNA damage (Saldivar et al., 2017). A number of small molecules have now been described that demonstrate potent specificity for the inhibition of the ATR kinase (Charrier et al., 2011; Foote et al., 2018; Wengner et al., 2019). The availability of ATR inhibitors has led to a number of preclinical studies in a variety of disease models and subsequent clinical testing (Leszczynska et al., 2016; Dillon et al., 2019; Vendetti et al., 2018; Pires et al., 2012). Many downstream targets of ATR have been described, including the checkpoint kinase Chk1, some of which are also potential biomarkers.In addition to decreased phosphorylation of characterized downstream targets, ATR inhibitors have been demonstrated to have a number of less obvious effects such as impacting the transcription of specific genes, including an increase in genes involved in senescence-associated secretory pathways (SASP) and endoplasmic reticulum (ER) stress. For example, ATR inhibition (using VE-821) was found to lead to increased p62 expression at both the mRNA and protein levels (Muralidharan et al., 2016). p62 is also known as sequestosome 1 (SQSTM1), which is reflective of its role as a ubiquitin-binding scaffold protein that targets proteins for selective autophagy (Zaffagnini et al., 2018). Autophagy is a highly conserved cytoprotective process that allows the recycling of cellular components in response to stress and is often dysregulated in cancer (Dikic and Elazar, 2018). The loss of p62 expression is a commonly used marker of autophagy and therefore an increase in p62 can be interpreted as a blockage in autophagy (Klionsky et al., 2016). However, the reported induction of p62 observed in response to the ATR inhibitor, VE-821, was not linked to a blockage in autophagy as another commonly used marker of autophagy, light chain 3 (LC3), appeared unaffected (Muralidharan et al., 2016). In a follow-on study focused on melanoma, inhibition of ATR was again demonstrated to lead to the induction of SASP/ER stress, which included an accumulation of p62 and the transcription factor C/EBP homologous protein (CHOP) (Muralidharan et al., 2017). The accumulation of p62 has also been implicated in compromising DNA repair, genomic integrity, and in accelerated aging (Eliopoulos et al., 2016; Hewitt et al., 2016; Kang et al., 2015; Kwon et al., 2012). A recent report demonstrated that accumulation of p62 hindered recruitment of factors including BRCA1, Rad51, and RAP80 to double-strand breaks leading to a decreased ability to repair damaged DNA (Wang et al., 2016). p62 also plays a role in both the DNA- and cGAMP-stimulated degradation of STING, which acts as a negative regulator of interferon signaling (Prabakaran et al., 2018). Interestingly, oxidation of p62 has recently been reported and described as a redox-sensitive mechanism linking oxidative stress and autophagy (Carroll et al., 2018; Otten et al., 2018).ATR inhibitors (AZD6738, VX-970/M6620, and BAY-1895344) are all currently being tested in a range of clinical trials (ClinicalTrials.gov). These include testing of VX-970 in a phase I dose escalation safety study in combination with chemoradiotherapy in esophageal and other solid cancers (EudraCT Number: 2015-003965-27). In this study, we set out to determine whether inhibition of ATR led to an accumulation of p62 in esophageal cell lines and to investigate the relevant mechanism and biological consequence.
Results
VX-970 Treatment Leads to p62 Accumulation
Esophageal cancer cells are sensitive to ATR inhibition although the extent of the response varies (Figure S1A) (Leszczynska et al., 2016). We investigated the expression of p62 protein in response to VX-970 treatment in four esophageal cell lines, including both squamous and adenocarcinoma cell lines. In each case a significant increase in p62 was observed after VX-970 treatment (Figure 1A). The accumulation of p62 in response to VX-970 was not restricted to esophageal cell lines and was also observed in a lung adenocarcinoma cell line, A549 (Figure S1B). The accumulation of p62 in response to VX-970 was found to be dose dependent and was evident from 0.1 μM (Figure S1C). Furthermore, we observed accumulation of p62 in response to VX-970 in three non-cancer cell lines, although this was less pronounced compared with the cancer cell lines (Figure 1B). To determine the cellular localization of p62 in VX-970 treated cells, we carried out both immunofluorescence analysis and cell fractionation. By microscopy, we saw significant accumulation of p62 predominantly in the cytoplasm of VX-970-treated OE21 cells with over 70% of the cells displaying increased p62 aggregates (Figures 1C and S1D). As a positive control for ATR inhibition, we also visualized γH2AX and found that 50% of the cells were positive for pan-nuclear ɣH2AX. Interestingly, cellular fractionation confirmed that both the cytoplasmic and nuclear expression of p62 increased (Figure 1D). To determine if p62 accumulation in these esophageal cell lines occurred via the same mechanism as previously reported, OE21 cells were treated with VX-970 in the absence of any other exogenous stress and qPCR carried out for the well-characterized target of ER stress; CHOP, as well as p62 (Muralidharan et al., 2016, 2017). Surprisingly and in contrast to the previous reports, neither the level of p62 nor CHOP mRNA was significantly altered in response to exposure to VX-970 (Figure 1E). To verify that CHOP is responsive to ER stress inducers, cells were also treated with thapsigargin or tunicamycin and a significant increase in CHOP was confirmed (Figure S1E). Together, these data demonstrate a significant accumulation of p62 protein in response to VX-970, which does not depend on increased mRNA levels of CHOP. Finally, we carried out immunohistochemistry on samples from a previous study (Leszczynska et al., 2016) to determine if p62 accumulated in OE-21 cells grown as xenograft tumors after treatment with VX-970. These data, although preliminary and limited by sample number (n = 3), suggest that p62 expression is higher in VX-970-treated tumors compared with untreated (Figure S1F).
Figure 1
VX-970 Treatment Leads to an Accumulation of p62
(A) The esophageal cell lines indicated were treated with VX-970 (1 μM) for the times shown. Western blotting was carried out for the detection of p62 expression. β-Actin was used as a loading control. OE21 (n = 3), OE33 (n = 2), FLO-1 (n = 2), KYSE (n = 1).
(B) MRC5, HFL1, and RPE cells were exposed to 1 μM VX-970 for 14 h followed by western blotting as indicated. β-Actin was used as a loading control. MRC-5 (n = 2), HFL1/RPE (n = 1).
(C) OE21 cells were exposed to 1 μM VX-970 for 8 and 18 h followed by staining for p62 (green) or ɣH2AX (green). DAPI (blue) was used to visualize the nucleus (n = 2).
(D) OE21 cells were exposed to VX-970 (1 μM) for 18 h and subjected to cellular fractionation to produce cytoplasmic and nuclear fractions. Whole-cell extracts (WCE) were included. α-Tubulin and Lamin-B1 were used as cytoplasmic and nuclear controls, respectively (n = 3).
(E) OE21 cells were exposed to VX-970 (1 μM) for the indicated times. qPCR was carried out to determine mRNA levels for p62 and CHOP, which were normalized to 18S mRNA. Data represent fold change in gene expression relative to DMSO control (0 h). Means ± SD of three independent experiments are shown.
VX-970 Treatment Leads to an Accumulation of p62(A) The esophageal cell lines indicated were treated with VX-970 (1 μM) for the times shown. Western blotting was carried out for the detection of p62 expression. β-Actin was used as a loading control. OE21 (n = 3), OE33 (n = 2), FLO-1 (n = 2), KYSE (n = 1).(B) MRC5, HFL1, and RPE cells were exposed to 1 μM VX-970 for 14 h followed by western blotting as indicated. β-Actin was used as a loading control. MRC-5 (n = 2), HFL1/RPE (n = 1).(C) OE21 cells were exposed to 1 μM VX-970 for 8 and 18 h followed by staining for p62 (green) or ɣH2AX (green). DAPI (blue) was used to visualize the nucleus (n = 2).(D) OE21 cells were exposed to VX-970 (1 μM) for 18 h and subjected to cellular fractionation to produce cytoplasmic and nuclear fractions. Whole-cell extracts (WCE) were included. α-Tubulin and Lamin-B1 were used as cytoplasmic and nuclear controls, respectively (n = 3).(E) OE21 cells were exposed to VX-970 (1 μM) for the indicated times. qPCR was carried out to determine mRNA levels for p62 and CHOP, which were normalized to 18S mRNA. Data represent fold change in gene expression relative to DMSO control (0 h). Means ± SD of three independent experiments are shown.
VX-970 Leads to p62 Accumulation in an ATR-Independent Manner
Next, we sought to verify that the accumulation of p62 in response to VX-970 was the result of ATR inhibition. To achieve this, OE21 cells were exposed to alternative ATR inhibitors (VE-821, AZD6738, or BAY-1895344) and p62 levels were determined. Like VX-970 and as previously reported for other cancer types, exposure to the structurally related VE-821 led to an accumulation of p62 (Figure 2A). However, in contrast to treatment with VX-970 and VE-821, we saw no accumulation of p62 in cells treated with various concentrations of either AZD6738 or BAY-1895344 (Figures 2B and 2C), which are structurally distinct to VE-821 and VX-970. As expected, in response to all the ATR inhibitors γH2AX increased, indicative of ATR inhibition (Figures 2B–2D and S2A). To rule out the possibility that the kinetics of p62 accumulation in response to AZD6738 or BAY-1895344 differed to those observed for VX-970, a time course was carried out. No accumulation of p62 was observed in response to AZD6738 or BAY-1895344 over a 24-h period (Figures S2B and S2C). These data suggest that the p62 accumulation in response to VX-970/VE-821 occurs independently of ATR inhibition. This was supported by the finding that inhibition of Chk1, a key downstream target of ATR, did not lead to accumulation of p62 (Figure S2D). To further test this hypothesis, we analyzed the levels of p62 in ATR heterozygous HCT116 cells, which express significantly less ATR protein than the matched controls. The baseline level of p62 in these cells was similar despite the difference in ATR, and in both cases, when VX-970 was added an increase in p62 was observed (Figure 2D). Next, we used cells from a patient with Seckel syndrome and a normal matched control. In this case the levels of p62 did differ between the two cell lines; however, it was the ATR proficient cell line that had the highest level of p62 (Figure 2E). Finally, we used siRNA to deplete ATR and determined the levels of p62. The siRNA treatment led to an almost complete loss of detectable ATR protein, but this had no impact on the levels of p62 (Figure 2F). Together, these data demonstrate that, although VE-821 and VX-970 led to an accumulation of p62, this is not due to inhibition of ATR. Kinase inhibitors are highly likely to inhibit related kinases to some extent and in the case of ATR inhibitors these are most likely ATM and DNA-pk. Although less significant, weak inhibition of mTOR has also been reported for some ATR inhibitors (Figure S3A). Inhibition of mTOR is predicted to lead to increased autophagy and reduced p62 and so is unlikely to explain the mechanism of p62 induction observed in response to VE-821 and VX-970 (Kakiuchi et al., 2019; Villar et al., 2017). The structures of the four ATR inhibitors we have investigated are shown (Figure 3). We considered whether the chemical properties of the compounds, including the predicted pKa, protonation states (at pH 7.4), and cLog D values of the inhibitors correlate with the ability to induce p62. Although we found no such relationship (Figures S3B–S3E), we note that VE-821 and VX-970 are structurally related and both incorporate the pyrazin-2-amine motif. AZD6738 and BAY-1895344 both possess an (R)-3-methylmorpholino group and are structurally distinct to VE-821 and VX-970.
Figure 2
Accumulation of p62 is Not Dependent on Inhibition of ATR
(A) OE21 cells were exposed to 1 μM VE-821 for the time points indicated (n = 3).
(B) OE21 cells were exposed to the range of doses indicated of AZD6738 for 16 h (n = 3).
(C) OE21 cells were treated with BAY-1895344 for a period of 16 h using the doses indicated (n = 2). In (A)–(C) as a positive control, cells were treated with 1 μM VX-970 for 16 h. Quantification of p62 expression normalized to β-actin (p62/β-actin) is shown as a fold change below the loading controls. γH2AX was used as a marker for DNA damage.
(D) HCT116 ATRwt and HCT116 ATR−/flox cells were treated with 1 μM VX-970 for 16 h followed by immunoblotting for the indicated proteins (n = 2).
(E) Lymphoblastoid cells from a patient with Seckel syndrome and a normal matched control were exposed to VX-970 (1 μM) for 16 h followed by immunoblotting for the indicated proteins (n = 2).
(F) OE21 cells were untreated (UT) or transfected with either ATR siRNA (siATR) or mock control (Mock). Cells were harvested at 12, 24, and 36 h post transfection and immunoblotting was carried out with the indicated antibodies (n = 3). β-Actin was used as a loading control for all western blots.
Figure 3
The Chemical Structures of the ATR Inhibitors VE-821, VX-970, AZD6738, and BAY-1895344
Accumulation of p62 is Not Dependent on Inhibition of ATR(A) OE21 cells were exposed to 1 μM VE-821 for the time points indicated (n = 3).(B) OE21 cells were exposed to the range of doses indicated of AZD6738 for 16 h (n = 3).(C) OE21 cells were treated with BAY-1895344 for a period of 16 h using the doses indicated (n = 2). In (A)–(C) as a positive control, cells were treated with 1 μM VX-970 for 16 h. Quantification of p62 expression normalized to β-actin (p62/β-actin) is shown as a fold change below the loading controls. γH2AX was used as a marker for DNA damage.(D) HCT116 ATRwt and HCT116ATR−/flox cells were treated with 1 μM VX-970 for 16 h followed by immunoblotting for the indicated proteins (n = 2).(E) Lymphoblastoid cells from a patient with Seckel syndrome and a normal matched control were exposed to VX-970 (1 μM) for 16 h followed by immunoblotting for the indicated proteins (n = 2).(F) OE21 cells were untreated (UT) or transfected with either ATR siRNA (siATR) or mock control (Mock). Cells were harvested at 12, 24, and 36 h post transfection and immunoblotting was carried out with the indicated antibodies (n = 3). β-Actin was used as a loading control for all western blots.The Chemical Structures of the ATR Inhibitors VE-821, VX-970, AZD6738, and BAY-1895344
p62 Accumulates in a Redox-Independent Manner in Response to VX-970
Previous reports have determined that p62 levels increase in response to oxidative stress (Carroll et al., 2018). To test the hypothesis that VX-970-induced p62 might be the result of oxidative stress, we exposed OE21 cells to VX-970 in the presence of the ROS scavenger, N-acetyl-l-cysteine (NAC), and determined the impact on p62 accumulation. As expected, p62 and γH2AX accumulated in response to VX-970; however, the addition of NAC abrogated the induction of p62 and significantly reduced γH2AX (Figure 4A). This suggested that VX-970-mediated accumulation of p62 and DNA damage might be the result of increased ROS. The redox sensitivity of p62 was previously attributed to the formation of disulfide bonds between specific cysteine residues (105, 113) (Carroll et al., 2018). Using mouse embryonic fibroblasts (mefs) with these cysteines mutated to alanines, we asked if this mechanism was critical to the VX-970-dependent accumulation of p62. The matched mefs were exposed to VX-970 for up to 16 h followed by western blotting for p62. Preliminary results showed that VX-970 still led to p62 accumulation even in the mutant mefs, suggesting that p62 accumulation was independent of ROS (Figure S4A). To confirm whether VX-970 led to increased cellular ROS, we exposed OE21 cells to the ATR inhibitors and measured mitochondrial ROS using MitoSox after a period of 6 h (Figures 4B and S4B–S4D). None of the four ATR inhibitors tested (VX-970, VE-821, AZD6738, or BAY-1895344) significantly induced ROS during this time frame. This finding was supported with a subsequent DCFDAROS assay, which was undertaken in order to assess total cellular ROS levels, and again showed that the ATR inhibitors did not induce ROS in the time frame in which p62 accumulates (Figure 4C). Notably, longer exposure to the ATR inhibitors, including those that do not lead to p62 accumulation, resulted in the accumulation of mitochondrial ROS (Figures S5A–S5C). As expected, treatment with H2O2 led to a significant increase in ROS, and therefore, we asked if p62 accumulated in these conditions. OE21 cells were exposed to H2O2 but did not accumulate p62 (Figure 4D). Together, these data indicate that p62 is accumulating in a redox-independent manner and suggests that VX-970 leads to blockage in autophagy and subsequent p62 accumulation. To begin investigating this, we treated OE21 cells with chloroquine (CQ), a known autophagy blocker. As expected, treatment with CQ also led to p62 accumulation and surprisingly this too was inhibited by NAC treatment (Figure 4E). This finding is unexplained, although we did verify that CQ did not lead to increased ROS levels and so the effect on p62 accumulation is likely ROS independent (Figures S5D and S5E) and more likely dependent on the known non-specific activities of NAC (Halasi et al., 2013; Sun, 2010).
Figure 4
ROS Dependence of p62 Accumulation
(A) OE21 cells were treated with either VX-970 (1 μM) alone or co-treated with NAC (20 mM) at the time points indicated. Western blotting was carried out using the antibodies indicated. γH2AX was used to detect double-stranded breaks and β-actin was used as a loading control (n = 3).
(B) OE21 cells were treated with ATR inhibitors for 6 h as follows: VX-970 (1 μM), VE-821 (1 μM), AZD6738 (1 μM), or BAY-1895344 (20 nM). Cells were stained with MitoSox (5 μM) and assayed by flow cytometry. Tert-butyl hydroperoxide (TBHP) (150 mM) was used as positive control for ROS generation (p < 0.001 = ∗∗∗) (n = 3).
(C) OE21 cells were treated with ATR inhibitors, VE-821 (1 μM), AZD6738 (1 μM) or VX-970 (1 μM), for 8 h and ROS production was detected using the DCFDA ROS assay. H2O2 (100 μM) and TBHP (150 mM) were used as positive controls for ROS generation (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001) (n = 3).
(D) OE21 cells were treated with H2O2 (1 mM) at varying time points indicated and p62 protein expression was determined. VX-970 (1 μM, 18 h) was used as a positive control (n = 2). γH2AX was used as a control for ATR inhibition and β-actin was used as a loading control.
(E) OE21 cells were exposed to chloroquine (CQ, 30 μM) and/or NAC (20 mM) for 6 h. The levels of p62, LC3, and β-actin are shown (n = 2).
ROS Dependence of p62 Accumulation(A) OE21 cells were treated with either VX-970 (1 μM) alone or co-treated with NAC (20 mM) at the time points indicated. Western blotting was carried out using the antibodies indicated. γH2AX was used to detect double-stranded breaks and β-actin was used as a loading control (n = 3).(B) OE21 cells were treated with ATR inhibitors for 6 h as follows: VX-970 (1 μM), VE-821 (1 μM), AZD6738 (1 μM), or BAY-1895344 (20 nM). Cells were stained with MitoSox (5 μM) and assayed by flow cytometry. Tert-butyl hydroperoxide (TBHP) (150 mM) was used as positive control for ROS generation (p < 0.001 = ∗∗∗) (n = 3).(C) OE21 cells were treated with ATR inhibitors, VE-821 (1 μM), AZD6738 (1 μM) or VX-970 (1 μM), for 8 h and ROS production was detected using the DCFDAROS assay. H2O2 (100 μM) and TBHP (150 mM) were used as positive controls for ROS generation (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001) (n = 3).(D) OE21 cells were treated with H2O2 (1 mM) at varying time points indicated and p62 protein expression was determined. VX-970 (1 μM, 18 h) was used as a positive control (n = 2). γH2AX was used as a control for ATR inhibition and β-actin was used as a loading control.(E) OE21 cells were exposed to chloroquine (CQ, 30 μM) and/or NAC (20 mM) for 6 h. The levels of p62, LC3, and β-actin are shown (n = 2).
VX-970 Leads to a Blockage in Autophagy
Cells exposed to VX-970 rapidly underwent cytoplasmic vacuolization demonstrated by the presence of translucent vesicles (Figure 5A). Cytoplasmic vacuolization has recently been reported in response to VX-970 in metastatic osteosarcoma cells where it was linked to eventual cell death but not autophagy (Li et al., 2020). However, we further investigated the hypothesis that VX-970 may block autophagy. During autophagy, LC3-I is lipidated to form LC3-II, which is required for forming fully functional autophagosomes. Therefore, LC3 is commonly used as a marker for autophagy (Klionsky et al., 2016). OE21 cells were exposed to VX-970, and the levels of LC3 were determined to investigate changes in baseline autophagy rates. As shown previously, the levels of p62 increased with time and both LC3-I and II accumulated, suggesting that autophagy was indeed blocked (Figure 5B). No changes in LC3 were observed after exposure to VE-821, AZD6738, or BAY-1895344 (Figures S6A–S6C). These data demonstrate that, although both VX-970 and VE-821 lead to an accumulation of p62, only VX-970 also impacts LC3. Next, OE21 cells were exposed to VX-970 in hypoxic conditions of <0.1% oxygen, which has been shown to induce autophagy (Rzymski et al., 2010; Skwarska et al., 2017). As expected, exposure to hypoxia led to increased LC3-II and a decrease in p62. However, in the presence of VX-970 in hypoxia, the expression of LC3-II was further increased and p62 accumulated (Figure S6D). Again, this was not restricted to OE21 cells and was also observed in HCT116 and RKO cells (Figures S6E and S6F). This again suggests that autophagy flux is blocked by VX-970. Given that increased levels of LC3-II may indicate either increased formation of autophagosomes or blockage of autophagosome degradation, we next determined the effect of VX-970 on the autophagic flux in the presence of autophagy inhibitors. Autophagy is a multistep process, and a number of autophagy modulators are in common use that block autophagy at different stages, such as 3-methyladenine (3-MA), which blocks the early stage by suppressing the formation of autophagosomes; CQ and bafilomycin A1 (BafA1), which both block late stage autophagy through preventing fusion of the autophagosomes and lysosomes (Klionsky et al., 2016). If VX-970 alone increases the level of LC3-II, but there is no further increase in the presence of the autophagy inhibitors, then it is likely that autophagy flux is inhibited by VX-970 and autophagosomes do not undergo autophagic degradation. Co-treatment of OE21 cells with 3-MA and VX-970 abrogated the p62 accumulation observed with VX-970 treatment alone (Figure 5C). These data indicate that blocking autophagy at an early stage prevented the accumulation of p62 suggesting that the VX-970-mediated effect on autophagy occurred at a later stage. Treatment with either BafA1 or CQ alone led to the expected increase in p62 confirming that these agents block autophagy in OE21 cells. However, when BafA1 or CQ were used in combination with VX-970 no further increase in p62 was observed (Figures 5D and 5E). Together, these data indicate that, like BafA1 and CQ, VX-970 blocks late-stage autophagy by inhibiting autophagy flux.
Figure 5
VX-970 Treatment Leads to a Late-Stage Blockage in Autophagy
(A) OE21 cells were treated with VX-970 (1 μM), VE-821 (1 μM), AZD-6738 (1 μM), or BAY-1895344 (20 nM) for 8 h. Light microscopy demonstrates the presence of translucent vesicle (black arrow) in the live cells (n = 3). Images were taken using 20x objective.
(B) OE21 cells were exposed to VX-970 (1 μM) for the times indicated followed by western blotting. The levels of p62, LC3, and β-actin are shown (n = 3).
(C) OE21 cells were exposed to 3-methyladenine (3-MA, 10 mM), VX-970 (1 μM), or 3-MA + VX-970 for 6 h followed by western blotting. The levels of p62, LC3, and β-actin are shown (n = 3).
(D) OE21 cells were treated with bafilomycin A1 (BafA1, 100 nM), VX-970 (1 μM), or BafA1 + VX-970 (1 μM) for 6 h followed by western blotting. The levels of p62, LC3, and β-actin are shown (n = 3).
(E) OE21 cells were treated with VX-970 (1 μM), CQ (100 μM), or VX-970 + CQ for 6 h. CQ was added for 1 h before the end of VX-970 treatment. Western blotting was then carried out for p62, LC3, and β-actin (n = 2).
VX-970 Treatment Leads to a Late-Stage Blockage in Autophagy(A) OE21 cells were treated with VX-970 (1 μM), VE-821 (1 μM), AZD-6738 (1 μM), or BAY-1895344 (20 nM) for 8 h. Light microscopy demonstrates the presence of translucent vesicle (black arrow) in the live cells (n = 3). Images were taken using 20x objective.(B) OE21 cells were exposed to VX-970 (1 μM) for the times indicated followed by western blotting. The levels of p62, LC3, and β-actin are shown (n = 3).(C) OE21 cells were exposed to 3-methyladenine (3-MA, 10 mM), VX-970 (1 μM), or 3-MA + VX-970 for 6 h followed by western blotting. The levels of p62, LC3, and β-actin are shown (n = 3).(D) OE21 cells were treated with bafilomycin A1 (BafA1, 100 nM), VX-970 (1 μM), or BafA1 + VX-970 (1 μM) for 6 h followed by western blotting. The levels of p62, LC3, and β-actin are shown (n = 3).(E) OE21 cells were treated with VX-970 (1 μM), CQ (100 μM), or VX-970 + CQ for 6 h. CQ was added for 1 h before the end of VX-970 treatment. Western blotting was then carried out for p62, LC3, and β-actin (n = 2).
Discussion
This study has shown that induction of p62 protein by the ATR inhibitor, VX-970, occurs in an ATR-independent manner. This conclusion is based on evidence that an increase in p62 is attributed to only VX-970 and its precursor VE-821, whereas ATR inhibitors AZD6738 and BAY-1895344 have no effect on p62 levels. Furthermore, p62 expression was unaffected or decreased in ATR-deficient cell lines when compared with matched controls, and siRNA knockdown of ATR had no effect on p62 levels. It is possible that both VE-821 and VX-970 have the same off-target effect mediated through their shared structural features, whereas AZD6738 and BAY-1895344 do not share these features and hence do not evoke the same biological effects. Future work is required to conclusively determine whether these findings translate to an in vivo setting. However, we present preliminary data in a limited dataset that indicates that VX-970-mediated accumulation of p62 is evident in vivo, suggesting that this could potentially be an informative biomarker in patients treated with VX-970.A previous report linked p62 accumulation to oxidative stress; however, in our study we saw no evidence of increased ROS in response to VX-970 (Carroll et al., 2018). Somewhat confusingly, p62 accumulation upon VX-970 treatment was rescued by NAC, a ROS scavenger. NAC has been shown to have alternative functions in addition to a role as a ROS scavenger (Halasi et al., 2013; Sun, 2010). Therefore, it is probable that the abrogation of p62 induction by NAC is through another means and not due to its ROS scavenging function. Notably, p62 accumulation was also prevented by NAC in CQ-treated cells suggesting this is a general off-target effect. We have not pursued the use of alternative ROS scavengers instead of NAC as we have demonstrated that VX-970 does not increase ROS in the time frame in which p62 accumulates.Our findings agree with previous studies, which describe an induction of p62 in response to ATR inhibition using VE-821 (Muralidharan et al., 2016, 2017). In contrast to these reports, we have not been able to link inhibition of ATR to an increase in ER stress as we saw no increase in CHOP expression; it is possible that this difference reflects a cell type-specific response. However, in agreement with the previous reports, we also concluded that, despite the accumulation of p62 in response to VE-821, there was no significant blockage in autophagy, as determined by LC3. Notably, the p62 accumulation after VE-821 treatment was not as robust as after VX-970, leading us to conclude that, if the off-target effect of VX-970 and VE-821 is the same, it is less potent with VE-821.Based on the appearance of VX-970-treated cells, it seems that p62 is forming large aggregates or possibly p62 bodies that are either not targeted to the lysosome or are not being degraded (You et al., 2019). The consequence of increased p62 and/or blocked autophagy are difficult to predict and will undoubtedly be context dependent. However, autophagy is reported to be overactive in cancers and thought to play a role in tumor cell survival (Kimmelman and White, 2017) and an accumulation of p62 has been linked to a reduction in the repair of double-stranded DNA breaks (Wang et al., 2016). Therefore, it is possible that the additional property of inhibiting autophagy may increase the efficacy of VX-970 in clinical studies. Most importantly, this study highlights that caution is warranted when using pharmacological inhibitors as significant off-target effects can occur.
Limitations of the Study
Our data using specific inhibitors to block different stages of autophagy suggest that VX-970 inhibits the later stages of autophagy. However, further assays including genetically blocking autophagosome initiation and using a mRFP-GFP-LC3 tandem fluorescent probe to study autophagic flux would be required to fully investigate the nature of the VX-970-mediated autophagy block (Yoshii and Mizushima, 2017; Klionsky et al., 2016). Further investigation is required to address important questions including how the accumulation of p62 and blockage of autophagy influence cell fate.
Resource Availability
Lead Contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Ester Hammond (ester.hammond@oncology.ox.ac.uk).
Materials Availability
This study did not generate new unique reagents.
Data and Code Availability
This study did not generate/analyze datasets/code.
Methods
All methods can be found in the accompanying Transparent Methods supplemental file.
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Shailendra Anoopkumar-Dukie; Manuela Antonioli; Hiroshi Aoki; Nadezda Apostolova; Saveria Aquila; Katia Aquilano; Koichi Araki; Eli Arama; Agustin Aranda; Jun Araya; Alexandre Arcaro; Esperanza Arias; Hirokazu Arimoto; Aileen R Ariosa; Jane L Armstrong; Thierry Arnould; Ivica Arsov; Katsuhiko Asanuma; Valerie Askanas; Eric Asselin; Ryuichiro Atarashi; Sally S Atherton; Julie D Atkin; Laura D Attardi; Patrick Auberger; Georg Auburger; Laure Aurelian; Riccardo Autelli; Laura Avagliano; Maria Laura Avantaggiati; Limor Avrahami; Suresh Awale; Neelam Azad; Tiziana Bachetti; Jonathan M Backer; Dong-Hun Bae; Jae-Sung Bae; Ok-Nam Bae; Soo Han Bae; Eric H Baehrecke; Seung-Hoon Baek; Stephen Baghdiguian; Agnieszka Bagniewska-Zadworna; Hua Bai; Jie Bai; Xue-Yuan Bai; Yannick Bailly; Kithiganahalli Narayanaswamy Balaji; Walter Balduini; Andrea Ballabio; Rena Balzan; Rajkumar Banerjee; Gábor Bánhegyi; Haijun Bao; Benoit Barbeau; Maria D Barrachina; Esther Barreiro; Bonnie Bartel; Alberto Bartolomé; Diane C Bassham; Maria Teresa Bassi; Robert C Bast; Alakananda Basu; Maria Teresa Batista; Henri Batoko; Maurizio Battino; Kyle Bauckman; Bradley L Baumgarner; K Ulrich Bayer; Rupert Beale; Jean-François Beaulieu; George R Beck; Christoph Becker; J David Beckham; Pierre-André Bédard; Patrick J Bednarski; Thomas J Begley; Christian Behl; Christian Behrends; Georg Mn Behrens; Kevin E Behrns; Eloy Bejarano; Amine Belaid; Francesca Belleudi; Giovanni Bénard; Guy Berchem; Daniele Bergamaschi; Matteo Bergami; Ben Berkhout; Laura Berliocchi; Amélie Bernard; Monique Bernard; Francesca Bernassola; Anne Bertolotti; Amanda S Bess; Sébastien Besteiro; Saverio Bettuzzi; Savita Bhalla; Shalmoli Bhattacharyya; Sujit K Bhutia; Caroline Biagosch; Michele Wolfe Bianchi; Martine Biard-Piechaczyk; Viktor Billes; Claudia Bincoletto; Baris Bingol; Sara W Bird; Marc Bitoun; Ivana Bjedov; Craig Blackstone; Lionel Blanc; Guillermo A Blanco; Heidi Kiil Blomhoff; Emilio Boada-Romero; Stefan Böckler; Marianne Boes; Kathleen Boesze-Battaglia; Lawrence H Boise; Alessandra Bolino; Andrea Boman; Paolo Bonaldo; Matteo Bordi; Jürgen Bosch; Luis M Botana; Joelle Botti; German Bou; Marina Bouché; Marion Bouchecareilh; Marie-Josée Boucher; Michael E Boulton; Sebastien G Bouret; Patricia Boya; Michaël Boyer-Guittaut; Peter V Bozhkov; Nathan Brady; Vania Mm Braga; Claudio Brancolini; Gerhard H Braus; José M Bravo-San Pedro; Lisa A Brennan; Emery H Bresnick; Patrick Brest; Dave Bridges; Marie-Agnès Bringer; Marisa Brini; Glauber C Brito; Bertha Brodin; Paul S Brookes; Eric J Brown; Karen Brown; Hal E Broxmeyer; Alain Bruhat; Patricia Chakur Brum; John H Brumell; Nicola Brunetti-Pierri; Robert J Bryson-Richardson; Shilpa Buch; Alastair M Buchan; Hikmet Budak; Dmitry V Bulavin; Scott J Bultman; Geert Bultynck; Vladimir Bumbasirevic; Yan Burelle; Robert E Burke; Margit Burmeister; Peter Bütikofer; Laura Caberlotto; Ken Cadwell; Monika Cahova; Dongsheng Cai; Jingjing Cai; Qian Cai; Sara Calatayud; Nadine Camougrand; Michelangelo Campanella; Grant R Campbell; Matthew Campbell; Silvia Campello; Robin Candau; Isabella Caniggia; Lavinia Cantoni; Lizhi Cao; Allan B Caplan; Michele Caraglia; Claudio Cardinali; Sandra Morais Cardoso; Jennifer S Carew; Laura A Carleton; Cathleen R Carlin; Silvia Carloni; Sven R Carlsson; Didac Carmona-Gutierrez; Leticia Am Carneiro; Oliana Carnevali; Serena Carra; Alice Carrier; Bernadette Carroll; Caty Casas; Josefina Casas; Giuliana Cassinelli; Perrine Castets; Susana Castro-Obregon; Gabriella Cavallini; Isabella Ceccherini; Francesco Cecconi; Arthur I Cederbaum; Valentín Ceña; Simone Cenci; Claudia Cerella; Davide Cervia; Silvia Cetrullo; Hassan Chaachouay; Han-Jung Chae; Andrei S Chagin; Chee-Yin Chai; Gopal Chakrabarti; Georgios Chamilos; Edmond Yw Chan; Matthew Tv Chan; Dhyan Chandra; Pallavi Chandra; Chih-Peng Chang; Raymond Chuen-Chung Chang; Ta Yuan Chang; John C Chatham; Saurabh Chatterjee; Santosh Chauhan; Yongsheng Che; Michael E Cheetham; Rajkumar Cheluvappa; Chun-Jung Chen; Gang Chen; Guang-Chao Chen; Guoqiang Chen; Hongzhuan Chen; Jeff W Chen; Jian-Kang Chen; Min Chen; Mingzhou Chen; Peiwen Chen; Qi Chen; Quan Chen; Shang-Der Chen; Si Chen; Steve S-L Chen; Wei Chen; Wei-Jung Chen; Wen Qiang Chen; Wenli Chen; Xiangmei Chen; Yau-Hung Chen; Ye-Guang Chen; Yin Chen; Yingyu Chen; Yongshun Chen; Yu-Jen Chen; Yue-Qin Chen; Yujie Chen; Zhen Chen; Zhong Chen; Alan Cheng; Christopher Hk Cheng; Hua Cheng; Heesun Cheong; Sara Cherry; Jason Chesney; Chun Hei Antonio Cheung; Eric Chevet; Hsiang Cheng Chi; Sung-Gil Chi; Fulvio Chiacchiera; Hui-Ling Chiang; Roberto Chiarelli; Mario Chiariello; Marcello Chieppa; Lih-Shen Chin; Mario Chiong; Gigi Nc Chiu; Dong-Hyung Cho; Ssang-Goo Cho; William C Cho; Yong-Yeon Cho; Young-Seok Cho; Augustine Mk Choi; Eui-Ju Choi; Eun-Kyoung Choi; Jayoung Choi; Mary E Choi; Seung-Il Choi; Tsui-Fen Chou; Salem Chouaib; Divaker Choubey; Vinay Choubey; Kuan-Chih Chow; Kamal Chowdhury; Charleen T Chu; Tsung-Hsien Chuang; Taehoon Chun; Hyewon Chung; Taijoon Chung; Yuen-Li Chung; Yong-Joon Chwae; Valentina Cianfanelli; Roberto Ciarcia; Iwona A Ciechomska; Maria Rosa Ciriolo; Mara Cirone; Sofie Claerhout; Michael J Clague; Joan Clària; Peter Gh Clarke; Robert Clarke; Emilio Clementi; Cédric Cleyrat; Miriam Cnop; Eliana M Coccia; Tiziana Cocco; Patrice Codogno; Jörn Coers; Ezra Ew Cohen; David Colecchia; Luisa Coletto; Núria S Coll; Emma Colucci-Guyon; Sergio Comincini; Maria Condello; Katherine L Cook; Graham H Coombs; Cynthia D Cooper; J Mark Cooper; Isabelle Coppens; Maria Tiziana Corasaniti; Marco Corazzari; Ramon Corbalan; Elisabeth Corcelle-Termeau; Mario D Cordero; Cristina Corral-Ramos; Olga Corti; Andrea Cossarizza; Paola Costelli; Safia Costes; Susan L Cotman; Ana Coto-Montes; Sandra Cottet; Eduardo Couve; Lori R Covey; L Ashley Cowart; Jeffery S Cox; Fraser P Coxon; Carolyn B Coyne; Mark S Cragg; Rolf J Craven; Tiziana Crepaldi; Jose L Crespo; Alfredo Criollo; Valeria Crippa; Maria Teresa Cruz; Ana Maria Cuervo; Jose M Cuezva; Taixing Cui; Pedro R Cutillas; Mark J Czaja; Maria F Czyzyk-Krzeska; Ruben K Dagda; Uta Dahmen; Chunsun Dai; Wenjie Dai; Yun Dai; Kevin N Dalby; Luisa Dalla Valle; Guillaume Dalmasso; Marcello D'Amelio; Markus Damme; Arlette Darfeuille-Michaud; Catherine Dargemont; Victor M Darley-Usmar; Srinivasan Dasarathy; Biplab Dasgupta; Srikanta Dash; Crispin R Dass; Hazel Marie Davey; Lester M Davids; David Dávila; Roger J Davis; Ted M Dawson; Valina L Dawson; Paula Daza; Jackie de Belleroche; Paul de Figueiredo; Regina Celia Bressan Queiroz de Figueiredo; José de la Fuente; Luisa De Martino; Antonella De Matteis; Guido Ry De Meyer; Angelo De Milito; Mauro De Santi; Wanderley de Souza; Vincenzo De Tata; Daniela De Zio; Jayanta Debnath; Reinhard Dechant; Jean-Paul Decuypere; Shane Deegan; Benjamin Dehay; Barbara Del Bello; Dominic P Del Re; Régis Delage-Mourroux; Lea Md Delbridge; Louise Deldicque; Elizabeth Delorme-Axford; Yizhen Deng; Joern Dengjel; Melanie Denizot; Paul Dent; Channing J Der; Vojo Deretic; Benoît Derrien; Eric Deutsch; Timothy P Devarenne; Rodney J Devenish; Sabrina Di Bartolomeo; Nicola Di Daniele; Fabio Di Domenico; Alessia Di Nardo; Simone Di Paola; Antonio Di Pietro; Livia Di Renzo; Aaron DiAntonio; Guillermo Díaz-Araya; Ines Díaz-Laviada; Maria T Diaz-Meco; Javier Diaz-Nido; Chad A Dickey; Robert C Dickson; Marc Diederich; Paul Digard; Ivan Dikic; Savithrama P Dinesh-Kumar; Chan Ding; Wen-Xing Ding; Zufeng Ding; Luciana Dini; Jörg Hw Distler; Abhinav Diwan; Mojgan Djavaheri-Mergny; Kostyantyn Dmytruk; Renwick Cj Dobson; Volker Doetsch; Karol Dokladny; Svetlana Dokudovskaya; Massimo Donadelli; X Charlie Dong; Xiaonan Dong; Zheng Dong; Terrence M Donohue; Kelly S Doran; Gabriella D'Orazi; Gerald W Dorn; Victor Dosenko; Sami Dridi; Liat Drucker; Jie Du; Li-Lin Du; Lihuan Du; André du Toit; Priyamvada Dua; Lei Duan; Pu Duann; Vikash Kumar Dubey; Michael R Duchen; Michel A Duchosal; Helene Duez; Isabelle Dugail; Verónica I Dumit; Mara C Duncan; Elaine A Dunlop; William A Dunn; Nicolas Dupont; Luc Dupuis; Raúl V Durán; Thomas M Durcan; Stéphane Duvezin-Caubet; Umamaheswar Duvvuri; Vinay Eapen; Darius Ebrahimi-Fakhari; Arnaud Echard; Leopold Eckhart; Charles L Edelstein; Aimee L Edinger; Ludwig Eichinger; Tobias Eisenberg; Avital Eisenberg-Lerner; N Tony Eissa; Wafik S El-Deiry; Victoria El-Khoury; Zvulun Elazar; Hagit Eldar-Finkelman; Chris Jh Elliott; Enzo Emanuele; Urban Emmenegger; Nikolai Engedal; Anna-Mart Engelbrecht; Simone Engelender; Jorrit M Enserink; Ralf Erdmann; Jekaterina Erenpreisa; Rajaraman Eri; Jason L Eriksen; Andreja Erman; Ricardo Escalante; Eeva-Liisa Eskelinen; Lucile Espert; Lorena Esteban-Martínez; Thomas J Evans; Mario Fabri; Gemma Fabrias; Cinzia Fabrizi; Antonio Facchiano; Nils J Færgeman; Alberto Faggioni; W Douglas Fairlie; Chunhai Fan; Daping Fan; Jie Fan; Shengyun Fang; Manolis Fanto; Alessandro Fanzani; Thomas Farkas; Mathias Faure; Francois B Favier; Howard Fearnhead; Massimo Federici; Erkang Fei; Tania C Felizardo; Hua Feng; Yibin Feng; Yuchen Feng; Thomas A Ferguson; Álvaro F Fernández; Maite G Fernandez-Barrena; Jose C Fernandez-Checa; Arsenio Fernández-López; Martin E Fernandez-Zapico; Olivier Feron; Elisabetta Ferraro; Carmen Veríssima Ferreira-Halder; Laszlo Fesus; Ralph Feuer; Fabienne C Fiesel; Eduardo C Filippi-Chiela; Giuseppe Filomeni; Gian Maria Fimia; John H Fingert; Steven Finkbeiner; Toren Finkel; Filomena Fiorito; Paul B Fisher; Marc Flajolet; Flavio Flamigni; Oliver Florey; Salvatore Florio; R Andres Floto; Marco Folini; Carlo Follo; Edward A Fon; Francesco Fornai; Franco Fortunato; Alessandro Fraldi; Rodrigo Franco; Arnaud Francois; Aurélie François; Lisa B Frankel; Iain Dc Fraser; Norbert Frey; Damien G Freyssenet; Christian Frezza; Scott L Friedman; Daniel E Frigo; Dongxu Fu; José M Fuentes; Juan Fueyo; Yoshio Fujitani; Yuuki Fujiwara; Mikihiro Fujiya; Mitsunori Fukuda; Simone Fulda; Carmela Fusco; Bozena Gabryel; Matthias Gaestel; Philippe Gailly; Malgorzata Gajewska; Sehamuddin Galadari; Gad Galili; Inmaculada Galindo; Maria F Galindo; Giovanna Galliciotti; Lorenzo Galluzzi; Luca Galluzzi; Vincent Galy; Noor Gammoh; Sam Gandy; Anand K Ganesan; Swamynathan Ganesan; Ian G Ganley; Monique Gannagé; Fen-Biao Gao; Feng Gao; Jian-Xin Gao; Lorena García Nannig; Eleonora García Véscovi; Marina Garcia-Macía; Carmen Garcia-Ruiz; Abhishek D Garg; Pramod Kumar Garg; Ricardo Gargini; Nils Christian Gassen; Damián Gatica; Evelina Gatti; Julie Gavard; Evripidis Gavathiotis; Liang Ge; Pengfei Ge; Shengfang Ge; Po-Wu Gean; Vania Gelmetti; Armando A Genazzani; Jiefei Geng; Pascal Genschik; Lisa Gerner; Jason E Gestwicki; David A Gewirtz; Saeid Ghavami; Eric Ghigo; Debabrata Ghosh; Anna Maria Giammarioli; Francesca Giampieri; Claudia Giampietri; Alexandra Giatromanolaki; Derrick J Gibbings; Lara Gibellini; Spencer B Gibson; Vanessa Ginet; Antonio Giordano; Flaviano Giorgini; Elisa Giovannetti; Stephen E Girardin; Suzana Gispert; Sandy Giuliano; Candece L Gladson; Alvaro Glavic; Martin Gleave; Nelly Godefroy; Robert M Gogal; Kuppan Gokulan; Gustavo H Goldman; Delia Goletti; Michael S Goligorsky; Aldrin V Gomes; Ligia C Gomes; Hernando Gomez; Candelaria Gomez-Manzano; Rubén Gómez-Sánchez; Dawit Ap Gonçalves; Ebru Goncu; Qingqiu Gong; Céline Gongora; Carlos B Gonzalez; Pedro Gonzalez-Alegre; Pilar Gonzalez-Cabo; Rosa Ana González-Polo; Ing Swie Goping; Carlos Gorbea; Nikolai V Gorbunov; Daphne R Goring; Adrienne M Gorman; Sharon M Gorski; Sandro Goruppi; Shino Goto-Yamada; Cecilia Gotor; Roberta A Gottlieb; Illana Gozes; Devrim Gozuacik; Yacine Graba; Martin Graef; Giovanna E Granato; Gary Dean Grant; Steven Grant; Giovanni Luca Gravina; Douglas R Green; Alexander Greenhough; Michael T Greenwood; Benedetto Grimaldi; Frédéric Gros; Charles Grose; Jean-Francois Groulx; Florian Gruber; Paolo Grumati; Tilman Grune; Jun-Lin Guan; Kun-Liang Guan; Barbara Guerra; Carlos Guillen; Kailash Gulshan; Jan Gunst; Chuanyong Guo; Lei Guo; Ming Guo; Wenjie Guo; Xu-Guang Guo; Andrea A Gust; Åsa B Gustafsson; Elaine Gutierrez; Maximiliano G Gutierrez; Ho-Shin Gwak; Albert Haas; James E Haber; Shinji Hadano; Monica Hagedorn; David R Hahn; Andrew J Halayko; Anne Hamacher-Brady; Kozo Hamada; Ahmed Hamai; Andrea Hamann; Maho Hamasaki; Isabelle Hamer; Qutayba Hamid; Ester M Hammond; Feng Han; Weidong Han; James T Handa; John A Hanover; Malene Hansen; Masaru Harada; Ljubica Harhaji-Trajkovic; J Wade Harper; Abdel Halim Harrath; Adrian L Harris; James Harris; Udo Hasler; Peter Hasselblatt; Kazuhisa Hasui; Robert G Hawley; Teresa S Hawley; Congcong He; Cynthia Y He; Fengtian He; Gu He; Rong-Rong He; Xian-Hui He; You-Wen He; Yu-Ying He; Joan K Heath; Marie-Josée Hébert; Robert A Heinzen; Gudmundur Vignir Helgason; Michael Hensel; Elizabeth P Henske; Chengtao Her; Paul K Herman; Agustín Hernández; Carlos Hernandez; Sonia Hernández-Tiedra; Claudio Hetz; P Robin Hiesinger; Katsumi Higaki; Sabine Hilfiker; Bradford G Hill; Joseph A Hill; William D Hill; Keisuke Hino; Daniel Hofius; Paul Hofman; Günter U Höglinger; Jörg Höhfeld; Marina K Holz; Yonggeun Hong; David A Hood; Jeroen Jm Hoozemans; Thorsten Hoppe; Chin Hsu; Chin-Yuan Hsu; Li-Chung Hsu; Dong Hu; Guochang Hu; Hong-Ming Hu; Hongbo Hu; Ming Chang Hu; Yu-Chen Hu; Zhuo-Wei Hu; Fang Hua; Ya Hua; Canhua Huang; Huey-Lan Huang; Kuo-How Huang; Kuo-Yang Huang; Shile Huang; Shiqian Huang; Wei-Pang Huang; Yi-Ran Huang; Yong Huang; Yunfei Huang; Tobias B Huber; Patricia Huebbe; Won-Ki Huh; Juha J Hulmi; Gang Min Hur; James H Hurley; Zvenyslava Husak; Sabah Na Hussain; Salik Hussain; Jung Jin Hwang; Seungmin Hwang; Thomas Is Hwang; Atsuhiro Ichihara; Yuzuru Imai; Carol Imbriano; Megumi Inomata; Takeshi Into; Valentina Iovane; Juan L Iovanna; Renato V Iozzo; Nancy Y Ip; Javier E Irazoqui; Pablo Iribarren; Yoshitaka Isaka; Aleksandra J Isakovic; Harry Ischiropoulos; Jeffrey S Isenberg; Mohammad Ishaq; Hiroyuki Ishida; Isao Ishii; Jane E Ishmael; Ciro Isidoro; Ken-Ichi Isobe; Erika Isono; Shohreh Issazadeh-Navikas; Koji Itahana; Eisuke Itakura; Andrei I Ivanov; Anand Krishnan V Iyer; José M Izquierdo; Yotaro Izumi; Valentina Izzo; Marja Jäättelä; Nadia Jaber; Daniel John Jackson; William T Jackson; Tony George Jacob; Thomas S Jacques; Chinnaswamy Jagannath; Ashish Jain; Nihar Ranjan Jana; Byoung Kuk Jang; Alkesh Jani; Bassam Janji; Paulo Roberto Jannig; Patric J Jansson; Steve Jean; Marina Jendrach; Ju-Hong Jeon; Niels Jessen; Eui-Bae Jeung; Kailiang Jia; Lijun Jia; Hong Jiang; Hongchi Jiang; Liwen Jiang; Teng Jiang; Xiaoyan Jiang; Xuejun Jiang; Xuejun Jiang; Ying Jiang; Yongjun Jiang; Alberto Jiménez; Cheng Jin; Hongchuan Jin; Lei Jin; Meiyan Jin; Shengkan Jin; Umesh Kumar Jinwal; Eun-Kyeong Jo; Terje Johansen; Daniel E Johnson; Gail Vw Johnson; James D Johnson; Eric Jonasch; Chris Jones; Leo Ab Joosten; Joaquin Jordan; Anna-Maria Joseph; Bertrand Joseph; Annie M Joubert; Dianwen Ju; Jingfang Ju; Hsueh-Fen Juan; Katrin Juenemann; Gábor Juhász; Hye Seung Jung; Jae U Jung; Yong-Keun Jung; Heinz Jungbluth; Matthew J Justice; Barry Jutten; Nadeem O Kaakoush; Kai Kaarniranta; Allen Kaasik; Tomohiro Kabuta; Bertrand Kaeffer; Katarina Kågedal; Alon Kahana; Shingo Kajimura; Or Kakhlon; Manjula Kalia; Dhan V Kalvakolanu; Yoshiaki Kamada; Konstantinos Kambas; Vitaliy O Kaminskyy; Harm H Kampinga; Mustapha Kandouz; Chanhee Kang; Rui Kang; Tae-Cheon Kang; Tomotake Kanki; Thirumala-Devi Kanneganti; Haruo Kanno; Anumantha G Kanthasamy; Marc Kantorow; Maria Kaparakis-Liaskos; Orsolya Kapuy; Vassiliki Karantza; Md Razaul Karim; Parimal Karmakar; Arthur Kaser; Susmita Kaushik; Thomas Kawula; A Murat Kaynar; Po-Yuan Ke; Zun-Ji Ke; John H Kehrl; Kate E Keller; Jongsook Kim Kemper; Anne K Kenworthy; Oliver Kepp; Andreas Kern; Santosh Kesari; David Kessel; Robin Ketteler; Isis do Carmo Kettelhut; Bilon Khambu; Muzamil Majid Khan; Vinoth Km Khandelwal; Sangeeta Khare; Juliann G Kiang; Amy A Kiger; Akio Kihara; Arianna L Kim; Cheol Hyeon Kim; Deok Ryong Kim; Do-Hyung Kim; Eung Kweon Kim; Hye Young Kim; Hyung-Ryong Kim; Jae-Sung Kim; Jeong Hun Kim; Jin Cheon Kim; Jin Hyoung Kim; Kwang Woon Kim; Michael D Kim; Moon-Moo Kim; Peter K Kim; Seong Who Kim; Soo-Youl Kim; Yong-Sun Kim; Yonghyun Kim; Adi Kimchi; Alec C Kimmelman; Tomonori Kimura; Jason S King; Karla Kirkegaard; Vladimir Kirkin; Lorrie A Kirshenbaum; Shuji Kishi; Yasuo Kitajima; Katsuhiko Kitamoto; Yasushi Kitaoka; Kaio Kitazato; Rudolf A Kley; Walter T Klimecki; Michael Klinkenberg; Jochen Klucken; Helene Knævelsrud; Erwin Knecht; Laura Knuppertz; Jiunn-Liang Ko; Satoru Kobayashi; Jan C Koch; Christelle Koechlin-Ramonatxo; Ulrich Koenig; Young Ho Koh; Katja Köhler; Sepp D Kohlwein; Masato Koike; Masaaki Komatsu; Eiki Kominami; Dexin Kong; Hee Jeong Kong; Eumorphia G Konstantakou; Benjamin T Kopp; Tamas Korcsmaros; Laura Korhonen; Viktor I Korolchuk; Nadya V Koshkina; Yanjun Kou; Michael I Koukourakis; Constantinos Koumenis; Attila L Kovács; Tibor Kovács; Werner J Kovacs; Daisuke Koya; Claudine Kraft; Dimitri Krainc; Helmut Kramer; Tamara Kravic-Stevovic; Wilhelm Krek; Carole Kretz-Remy; Roswitha Krick; Malathi Krishnamurthy; Janos Kriston-Vizi; Guido Kroemer; Michael C Kruer; Rejko Kruger; Nicholas T Ktistakis; Kazuyuki Kuchitsu; Christian Kuhn; Addanki Pratap Kumar; Anuj Kumar; Ashok Kumar; Deepak Kumar; Dhiraj Kumar; Rakesh Kumar; Sharad Kumar; Mondira Kundu; Hsing-Jien Kung; Atsushi Kuno; Sheng-Han Kuo; Jeff Kuret; Tino Kurz; Terry Kwok; Taeg Kyu Kwon; Yong Tae Kwon; Irene Kyrmizi; Albert R La Spada; Frank Lafont; Tim Lahm; Aparna Lakkaraju; Truong Lam; Trond Lamark; Steve Lancel; Terry H Landowski; Darius J R Lane; Jon D Lane; Cinzia Lanzi; Pierre Lapaquette; Louis R Lapierre; Jocelyn Laporte; Johanna Laukkarinen; Gordon W Laurie; Sergio Lavandero; Lena Lavie; Matthew J LaVoie; Betty Yuen Kwan Law; Helen Ka-Wai Law; Kelsey B Law; Robert Layfield; Pedro A Lazo; Laurent Le Cam; Karine G Le Roch; Hervé Le Stunff; Vijittra Leardkamolkarn; Marc Lecuit; Byung-Hoon Lee; Che-Hsin Lee; Erinna F Lee; Gyun Min Lee; He-Jin Lee; Hsinyu Lee; Jae Keun Lee; Jongdae Lee; Ju-Hyun Lee; Jun Hee Lee; Michael Lee; Myung-Shik Lee; Patty J Lee; Sam W Lee; Seung-Jae Lee; Shiow-Ju Lee; Stella Y Lee; Sug Hyung Lee; Sung Sik Lee; Sung-Joon Lee; Sunhee Lee; Ying-Ray Lee; Yong J Lee; Young H Lee; Christiaan Leeuwenburgh; Sylvain Lefort; Renaud Legouis; Jinzhi Lei; Qun-Ying Lei; David A Leib; Gil Leibowitz; Istvan Lekli; Stéphane D Lemaire; John J Lemasters; Marius K Lemberg; Antoinette Lemoine; Shuilong Leng; Guido Lenz; Paola Lenzi; Lilach O Lerman; Daniele Lettieri Barbato; Julia I-Ju Leu; Hing Y Leung; Beth Levine; Patrick A Lewis; Frank Lezoualc'h; Chi Li; Faqiang Li; Feng-Jun Li; Jun Li; Ke Li; Lian Li; Min Li; Min Li; Qiang Li; Rui Li; Sheng Li; Wei Li; Wei Li; Xiaotao Li; Yumin Li; Jiqin Lian; Chengyu Liang; Qiangrong Liang; Yulin Liao; Joana Liberal; Pawel P Liberski; Pearl Lie; Andrew P Lieberman; Hyunjung Jade Lim; Kah-Leong Lim; Kyu Lim; Raquel T Lima; Chang-Shen Lin; Chiou-Feng Lin; Fang Lin; Fangming Lin; Fu-Cheng Lin; Kui Lin; Kwang-Huei Lin; Pei-Hui Lin; Tianwei Lin; Wan-Wan Lin; Yee-Shin Lin; Yong Lin; Rafael Linden; Dan Lindholm; Lisa M Lindqvist; Paul Lingor; Andreas Linkermann; Lance A Liotta; Marta M Lipinski; Vitor A Lira; Michael P Lisanti; Paloma B Liton; Bo Liu; Chong Liu; Chun-Feng Liu; Fei Liu; Hung-Jen Liu; Jianxun Liu; Jing-Jing Liu; Jing-Lan Liu; Ke Liu; Leyuan Liu; Liang Liu; Quentin Liu; Rong-Yu Liu; Shiming Liu; Shuwen Liu; Wei Liu; Xian-De Liu; Xiangguo Liu; Xiao-Hong Liu; Xinfeng Liu; Xu Liu; Xueqin Liu; Yang Liu; Yule Liu; Zexian Liu; Zhe Liu; Juan P Liuzzi; Gérard Lizard; Mila Ljujic; Irfan J Lodhi; Susan E Logue; Bal L Lokeshwar; Yun Chau Long; Sagar Lonial; Benjamin Loos; Carlos López-Otín; Cristina López-Vicario; Mar Lorente; Philip L Lorenzi; Péter Lõrincz; Marek Los; Michael T Lotze; Penny E Lovat; Binfeng Lu; Bo Lu; Jiahong Lu; Qing Lu; She-Min Lu; Shuyan Lu; Yingying Lu; Frédéric Luciano; Shirley Luckhart; John Milton Lucocq; Paula Ludovico; Aurelia Lugea; Nicholas W Lukacs; Julian J Lum; Anders H Lund; Honglin Luo; Jia Luo; Shouqing Luo; Claudio Luparello; Timothy Lyons; Jianjie Ma; Yi Ma; Yong Ma; Zhenyi Ma; Juliano Machado; Glaucia M Machado-Santelli; Fernando Macian; Gustavo C MacIntosh; Jeffrey P MacKeigan; Kay F Macleod; John D MacMicking; Lee Ann MacMillan-Crow; Frank Madeo; Muniswamy Madesh; Julio Madrigal-Matute; Akiko Maeda; Tatsuya Maeda; Gustavo Maegawa; Emilia Maellaro; Hannelore Maes; Marta Magariños; Kenneth Maiese; Tapas K Maiti; Luigi Maiuri; Maria Chiara Maiuri; Carl G Maki; Roland Malli; Walter Malorni; Alina Maloyan; Fathia Mami-Chouaib; Na Man; Joseph D Mancias; Eva-Maria Mandelkow; Michael A Mandell; Angelo A Manfredi; Serge N Manié; Claudia Manzoni; Kai Mao; Zixu Mao; Zong-Wan Mao; Philippe Marambaud; Anna Maria Marconi; Zvonimir Marelja; Gabriella Marfe; Marta Margeta; Eva Margittai; Muriel Mari; Francesca V Mariani; Concepcio Marin; Sara Marinelli; Guillermo Mariño; Ivanka Markovic; Rebecca Marquez; Alberto M Martelli; Sascha Martens; Katie R Martin; Seamus J Martin; Shaun Martin; Miguel A Martin-Acebes; Paloma Martín-Sanz; Camille Martinand-Mari; Wim Martinet; Jennifer Martinez; Nuria Martinez-Lopez; Ubaldo Martinez-Outschoorn; Moisés Martínez-Velázquez; Marta Martinez-Vicente; Waleska Kerllen Martins; Hirosato Mashima; James A Mastrianni; Giuseppe Matarese; Paola Matarrese; Roberto Mateo; Satoaki Matoba; Naomichi Matsumoto; Takehiko Matsushita; Akira Matsuura; Takeshi Matsuzawa; Mark P Mattson; Soledad Matus; Norma Maugeri; Caroline Mauvezin; Andreas Mayer; Dusica Maysinger; Guillermo D Mazzolini; Mary Kate McBrayer; Kimberly McCall; Craig McCormick; Gerald M McInerney; Skye C McIver; Sharon McKenna; John J McMahon; Iain A McNeish; Fatima Mechta-Grigoriou; Jan Paul Medema; Diego L Medina; Klara Megyeri; Maryam Mehrpour; Jawahar L Mehta; Yide Mei; Ute-Christiane Meier; Alfred J Meijer; Alicia Meléndez; Gerry Melino; Sonia Melino; Edesio Jose Tenorio de Melo; Maria A Mena; Marc D Meneghini; Javier A Menendez; Regina Menezes; Liesu Meng; Ling-Hua Meng; Songshu Meng; Rossella Menghini; A Sue Menko; Rubem Fs Menna-Barreto; Manoj B Menon; Marco A Meraz-Ríos; Giuseppe Merla; Luciano Merlini; Angelica M Merlot; Andreas Meryk; Stefania Meschini; Joel N Meyer; Man-Tian Mi; Chao-Yu Miao; Lucia Micale; Simon Michaeli; Carine Michiels; Anna Rita Migliaccio; Anastasia Susie Mihailidou; Dalibor Mijaljica; Katsuhiko Mikoshiba; Enrico Milan; Leonor Miller-Fleming; Gordon B Mills; Ian G Mills; Georgia Minakaki; Berge A Minassian; Xiu-Fen Ming; Farida Minibayeva; Elena A Minina; Justine D Mintern; Saverio Minucci; Antonio Miranda-Vizuete; Claire H Mitchell; Shigeki Miyamoto; Keisuke Miyazawa; Noboru Mizushima; Katarzyna Mnich; Baharia Mograbi; Simin Mohseni; Luis Ferreira Moita; Marco Molinari; Maurizio Molinari; Andreas Buch Møller; Bertrand Mollereau; Faustino Mollinedo; Marco Mongillo; Martha M Monick; Serena Montagnaro; Craig Montell; Darren J Moore; Michael N Moore; Rodrigo Mora-Rodriguez; Paula I Moreira; Etienne Morel; Maria Beatrice Morelli; Sandra Moreno; Michael J Morgan; Arnaud Moris; Yuji Moriyasu; Janna L Morrison; Lynda A Morrison; Eugenia Morselli; Jorge Moscat; Pope L Moseley; Serge Mostowy; Elisa Motori; Denis Mottet; Jeremy C Mottram; Charbel E-H Moussa; Vassiliki E Mpakou; Hasan Mukhtar; Jean M Mulcahy Levy; Sylviane Muller; Raquel Muñoz-Moreno; Cristina Muñoz-Pinedo; Christian Münz; Maureen E Murphy; James T Murray; Aditya Murthy; Indira U Mysorekar; Ivan R Nabi; Massimo Nabissi; Gustavo A Nader; Yukitoshi Nagahara; Yoshitaka Nagai; Kazuhiro Nagata; Anika Nagelkerke; Péter Nagy; Samisubbu R Naidu; Sreejayan Nair; Hiroyasu Nakano; Hitoshi Nakatogawa; Meera Nanjundan; Gennaro Napolitano; Naweed I Naqvi; Roberta Nardacci; Derek P Narendra; Masashi Narita; Anna Chiara Nascimbeni; Ramesh Natarajan; Luiz C Navegantes; Steffan T Nawrocki; Taras Y Nazarko; Volodymyr Y Nazarko; Thomas Neill; Luca M Neri; Mihai G Netea; Romana T Netea-Maier; Bruno M Neves; Paul A Ney; Ioannis P Nezis; Hang Tt Nguyen; Huu Phuc Nguyen; Anne-Sophie Nicot; Hilde Nilsen; Per Nilsson; Mikio Nishimura; Ichizo Nishino; Mireia Niso-Santano; Hua Niu; Ralph A Nixon; Vincent Co Njar; Takeshi Noda; Angelika A Noegel; Elsie Magdalena Nolte; Erik Norberg; Koenraad K Norga; Sakineh Kazemi Noureini; Shoji Notomi; Lucia Notterpek; Karin Nowikovsky; Nobuyuki Nukina; Thorsten Nürnberger; Valerie B O'Donnell; Tracey O'Donovan; Peter J O'Dwyer; Ina Oehme; Clara L Oeste; Michinaga Ogawa; Besim Ogretmen; Yuji Ogura; Young J Oh; Masaki Ohmuraya; Takayuki Ohshima; Rani Ojha; Koji Okamoto; Toshiro Okazaki; F Javier Oliver; Karin Ollinger; Stefan Olsson; Daniel P Orban; Paulina Ordonez; Idil Orhon; Laszlo Orosz; Eyleen J O'Rourke; Helena Orozco; Angel L Ortega; Elena Ortona; Laura D Osellame; Junko Oshima; Shigeru Oshima; Heinz D Osiewacz; Takanobu Otomo; Kinya Otsu; Jing-Hsiung James Ou; Tiago F Outeiro; Dong-Yun Ouyang; Hongjiao Ouyang; Michael Overholtzer; Michelle A Ozbun; P Hande Ozdinler; Bulent Ozpolat; Consiglia Pacelli; Paolo Paganetti; Guylène Page; Gilles Pages; Ugo Pagnini; Beata Pajak; Stephen C Pak; Karolina Pakos-Zebrucka; Nazzy Pakpour; Zdena Palková; Francesca Palladino; Kathrin Pallauf; Nicolas Pallet; Marta Palmieri; Søren R Paludan; Camilla Palumbo; Silvia Palumbo; Olatz Pampliega; Hongming Pan; Wei Pan; Theocharis Panaretakis; Aseem Pandey; Areti Pantazopoulou; Zuzana Papackova; Daniela L Papademetrio; Issidora Papassideri; Alessio Papini; Nirmala Parajuli; Julian Pardo; Vrajesh V Parekh; Giancarlo Parenti; Jong-In Park; Junsoo Park; Ohkmae K Park; Roy Parker; Rosanna Parlato; Jan B Parys; Katherine R Parzych; Jean-Max Pasquet; Benoit Pasquier; Kishore Bs Pasumarthi; Daniel Patschan; Cam Patterson; Sophie Pattingre; Scott Pattison; Arnim Pause; Hermann Pavenstädt; Flaminia Pavone; Zully Pedrozo; Fernando J Peña; Miguel A Peñalva; Mario Pende; Jianxin Peng; Fabio Penna; Josef M Penninger; Anna Pensalfini; Salvatore Pepe; Gustavo Js Pereira; Paulo C Pereira; Verónica Pérez-de la Cruz; María Esther Pérez-Pérez; Diego Pérez-Rodríguez; Dolores Pérez-Sala; Celine Perier; Andras Perl; David H Perlmutter; Ida Perrotta; Shazib Pervaiz; Maija Pesonen; Jeffrey E Pessin; Godefridus J Peters; Morten Petersen; Irina Petrache; Basil J Petrof; Goran Petrovski; James M Phang; Mauro Piacentini; Marina Pierdominici; Philippe Pierre; Valérie Pierrefite-Carle; Federico Pietrocola; Felipe X Pimentel-Muiños; Mario Pinar; Benjamin Pineda; Ronit Pinkas-Kramarski; Marcello Pinti; Paolo Pinton; Bilal Piperdi; James M Piret; Leonidas C Platanias; Harald W Platta; Edward D Plowey; Stefanie Pöggeler; Marc Poirot; Peter Polčic; Angelo Poletti; Audrey H Poon; Hana Popelka; Blagovesta Popova; Izabela Poprawa; Shibu M Poulose; Joanna Poulton; Scott K Powers; Ted Powers; Mercedes Pozuelo-Rubio; Krisna Prak; Reinhild Prange; Mark Prescott; Muriel Priault; Sharon Prince; Richard L Proia; Tassula Proikas-Cezanne; Holger Prokisch; Vasilis J Promponas; Karin Przyklenk; Rosa Puertollano; Subbiah Pugazhenthi; Luigi Puglielli; Aurora Pujol; Julien Puyal; Dohun Pyeon; Xin Qi; Wen-Bin Qian; Zheng-Hong Qin; Yu Qiu; Ziwei Qu; Joe Quadrilatero; Frederick Quinn; Nina Raben; Hannah Rabinowich; Flavia Radogna; Michael J Ragusa; Mohamed Rahmani; Komal Raina; Sasanka Ramanadham; Rajagopal Ramesh; Abdelhaq Rami; Sarron Randall-Demllo; Felix Randow; Hai Rao; V Ashutosh Rao; Blake B Rasmussen; Tobias M Rasse; Edward A Ratovitski; Pierre-Emmanuel Rautou; Swapan K Ray; Babak Razani; Bruce H Reed; Fulvio Reggiori; Markus Rehm; Andreas S Reichert; Theo Rein; David J Reiner; Eric Reits; Jun Ren; Xingcong Ren; Maurizio Renna; Jane Eb Reusch; Jose L Revuelta; Leticia Reyes; Alireza R Rezaie; Robert I Richards; Des R Richardson; Clémence Richetta; Michael A Riehle; Bertrand H Rihn; Yasuko Rikihisa; Brigit E Riley; Gerald Rimbach; Maria Rita Rippo; Konstantinos Ritis; Federica Rizzi; Elizete Rizzo; Peter J Roach; Jeffrey Robbins; Michel Roberge; Gabriela Roca; Maria Carmela Roccheri; Sonia Rocha; Cecilia Mp Rodrigues; Clara I Rodríguez; Santiago Rodriguez de Cordoba; Natalia Rodriguez-Muela; Jeroen Roelofs; Vladimir V Rogov; Troy T Rohn; Bärbel Rohrer; Davide Romanelli; Luigina Romani; Patricia Silvia Romano; M Isabel G Roncero; Jose Luis Rosa; Alicia Rosello; Kirill V Rosen; Philip Rosenstiel; Magdalena Rost-Roszkowska; Kevin A Roth; Gael Roué; Mustapha Rouis; Kasper M Rouschop; Daniel T Ruan; Diego Ruano; David C Rubinsztein; Edmund B Rucker; Assaf Rudich; Emil Rudolf; Ruediger Rudolf; Markus A Ruegg; Carmen Ruiz-Roldan; Avnika Ashok Ruparelia; Paola Rusmini; David W Russ; Gian Luigi Russo; Giuseppe Russo; Rossella Russo; Tor Erik Rusten; Victoria Ryabovol; Kevin M Ryan; Stefan W Ryter; David M Sabatini; Michael Sacher; Carsten Sachse; Michael N Sack; Junichi Sadoshima; Paul Saftig; Ronit Sagi-Eisenberg; Sumit Sahni; Pothana Saikumar; Tsunenori Saito; Tatsuya Saitoh; Koichi Sakakura; Machiko Sakoh-Nakatogawa; Yasuhito Sakuraba; María Salazar-Roa; Paolo Salomoni; Ashok K Saluja; Paul M Salvaterra; Rosa Salvioli; Afshin Samali; Anthony Mj Sanchez; José A Sánchez-Alcázar; Ricardo Sanchez-Prieto; Marco Sandri; Miguel A Sanjuan; Stefano Santaguida; Laura Santambrogio; Giorgio Santoni; Claudia Nunes Dos Santos; Shweta Saran; Marco Sardiello; Graeme Sargent; Pallabi Sarkar; Sovan Sarkar; Maria Rosa Sarrias; Minnie M Sarwal; Chihiro Sasakawa; Motoko Sasaki; Miklos Sass; Ken Sato; Miyuki Sato; Joseph Satriano; Niramol Savaraj; Svetlana Saveljeva; Liliana Schaefer; Ulrich E Schaible; Michael Scharl; Hermann M Schatzl; Randy Schekman; Wiep Scheper; Alfonso Schiavi; Hyman M Schipper; Hana Schmeisser; Jens Schmidt; Ingo Schmitz; Bianca E Schneider; E Marion Schneider; Jaime L Schneider; Eric A Schon; Miriam J Schönenberger; Axel H Schönthal; Daniel F Schorderet; Bernd Schröder; Sebastian Schuck; Ryan J Schulze; Melanie Schwarten; Thomas L Schwarz; Sebastiano Sciarretta; Kathleen Scotto; A Ivana Scovassi; Robert A Screaton; Mark Screen; Hugo Seca; Simon Sedej; Laura Segatori; Nava Segev; Per O Seglen; Jose M Seguí-Simarro; Juan Segura-Aguilar; Ekihiro Seki; Christian Sell; Iban Seiliez; Clay F Semenkovich; Gregg L Semenza; Utpal Sen; Andreas L Serra; Ana Serrano-Puebla; Hiromi Sesaki; Takao Setoguchi; Carmine Settembre; John J Shacka; Ayesha N Shajahan-Haq; Irving M Shapiro; Shweta Sharma; Hua She; C-K James Shen; Chiung-Chyi Shen; Han-Ming Shen; Sanbing Shen; Weili Shen; Rui Sheng; Xianyong Sheng; Zu-Hang Sheng; Trevor G Shepherd; Junyan Shi; Qiang Shi; Qinghua Shi; Yuguang Shi; Shusaku Shibutani; Kenichi Shibuya; Yoshihiro Shidoji; Jeng-Jer Shieh; Chwen-Ming Shih; Yohta Shimada; Shigeomi Shimizu; Dong Wook Shin; Mari L Shinohara; Michiko Shintani; Takahiro Shintani; Tetsuo Shioi; Ken Shirabe; Ronit Shiri-Sverdlov; Orian Shirihai; Gordon C Shore; Chih-Wen Shu; Deepak Shukla; Andriy A Sibirny; Valentina Sica; Christina J Sigurdson; Einar M Sigurdsson; Puran Singh Sijwali; Beata Sikorska; Wilian A Silveira; Sandrine Silvente-Poirot; Gary A Silverman; Jan Simak; Thomas Simmet; Anna Katharina Simon; Hans-Uwe Simon; Cristiano Simone; Matias Simons; Anne Simonsen; Rajat Singh; Shivendra V Singh; Shrawan K Singh; Debasish Sinha; Sangita Sinha; Frank A Sinicrope; Agnieszka Sirko; Kapil Sirohi; Balindiwe Jn Sishi; Annie Sittler; Parco M Siu; Efthimios Sivridis; Anna Skwarska; Ruth Slack; Iva Slaninová; Nikolai Slavov; Soraya S Smaili; Keiran Sm Smalley; Duncan R Smith; Stefaan J Soenen; Scott A Soleimanpour; Anita Solhaug; Kumaravel Somasundaram; Jin H Son; Avinash Sonawane; Chunjuan Song; Fuyong Song; Hyun Kyu Song; Ju-Xian Song; Wei Song; Kai Y Soo; Anil K Sood; Tuck Wah Soong; Virawudh Soontornniyomkij; Maurizio Sorice; Federica Sotgia; David R Soto-Pantoja; Areechun Sotthibundhu; Maria João Sousa; Herman P Spaink; Paul N Span; Anne Spang; Janet D Sparks; Peter G Speck; Stephen A Spector; Claudia D Spies; Wolfdieter Springer; Daret St Clair; Alessandra Stacchiotti; Bart Staels; Michael T Stang; Daniel T Starczynowski; Petro Starokadomskyy; Clemens Steegborn; John W Steele; Leonidas Stefanis; Joan Steffan; Christine M Stellrecht; Harald Stenmark; Tomasz M Stepkowski; Stęphan T Stern; Craig Stevens; Brent R Stockwell; Veronika Stoka; Zuzana Storchova; Björn Stork; Vassilis Stratoulias; Dimitrios J Stravopodis; Pavel Strnad; Anne Marie Strohecker; Anna-Lena Ström; Per Stromhaug; Jiri Stulik; Yu-Xiong Su; Zhaoliang Su; Carlos S Subauste; Srinivasa Subramaniam; Carolyn M Sue; Sang Won Suh; Xinbing Sui; Supawadee Sukseree; David Sulzer; Fang-Lin Sun; Jiaren Sun; Jun Sun; Shi-Yong Sun; Yang Sun; Yi Sun; Yingjie Sun; Vinod Sundaramoorthy; Joseph Sung; Hidekazu Suzuki; Kuninori Suzuki; Naoki Suzuki; Tadashi Suzuki; Yuichiro J Suzuki; Michele S Swanson; Charles Swanton; Karl Swärd; Ghanshyam Swarup; Sean T Sweeney; Paul W Sylvester; Zsuzsanna Szatmari; Eva Szegezdi; Peter W Szlosarek; Heinrich Taegtmeyer; Marco Tafani; Emmanuel Taillebourg; Stephen Wg Tait; Krisztina Takacs-Vellai; Yoshinori Takahashi; Szabolcs Takáts; Genzou Takemura; Nagio Takigawa; Nicholas J Talbot; Elena Tamagno; Jerome Tamburini; Cai-Ping Tan; Lan Tan; Mei Lan Tan; Ming Tan; Yee-Joo Tan; Keiji Tanaka; Masaki Tanaka; Daolin Tang; Dingzhong Tang; Guomei Tang; Isei Tanida; Kunikazu Tanji; Bakhos A Tannous; Jose A Tapia; Inmaculada Tasset-Cuevas; Marc Tatar; Iman Tavassoly; Nektarios Tavernarakis; Allen Taylor; Graham S Taylor; Gregory A Taylor; J Paul Taylor; Mark J Taylor; Elena V Tchetina; Andrew R Tee; Fatima Teixeira-Clerc; Sucheta Telang; Tewin Tencomnao; Ba-Bie Teng; Ru-Jeng Teng; Faraj Terro; Gianluca Tettamanti; Arianne L Theiss; Anne E Theron; Kelly Jean Thomas; Marcos P Thomé; Paul G Thomes; Andrew Thorburn; Jeremy Thorner; Thomas Thum; Michael Thumm; Teresa Lm Thurston; Ling Tian; Andreas Till; Jenny Pan-Yun Ting; Vladimir I Titorenko; Lilach Toker; Stefano Toldo; Sharon A Tooze; Ivan Topisirovic; Maria Lyngaas Torgersen; Liliana Torosantucci; Alicia Torriglia; Maria Rosaria Torrisi; Cathy Tournier; Roberto Towns; Vladimir Trajkovic; Leonardo H Travassos; Gemma Triola; Durga Nand Tripathi; Daniela Trisciuoglio; Rodrigo Troncoso; Ioannis P Trougakos; Anita C Truttmann; Kuen-Jer Tsai; Mario P Tschan; Yi-Hsin Tseng; Takayuki Tsukuba; Allan Tsung; Andrey S Tsvetkov; Shuiping Tu; Hsing-Yu Tuan; Marco Tucci; David A Tumbarello; Boris Turk; Vito Turk; Robin Fb Turner; Anders A Tveita; Suresh C Tyagi; Makoto Ubukata; Yasuo Uchiyama; Andrej Udelnow; Takashi Ueno; Midori Umekawa; Rika Umemiya-Shirafuji; Benjamin R Underwood; Christian Ungermann; Rodrigo P Ureshino; Ryo Ushioda; Vladimir N Uversky; Néstor L Uzcátegui; Thomas Vaccari; Maria I Vaccaro; Libuše Váchová; Helin Vakifahmetoglu-Norberg; Rut Valdor; Enza Maria Valente; Francois Vallette; Angela M Valverde; Greet Van den Berghe; Ludo Van Den Bosch; Gijs R van den Brink; F Gisou van der Goot; Ida J van der Klei; Luc Jw van der Laan; Wouter G van Doorn; Marjolein van Egmond; Kenneth L van Golen; Luc Van Kaer; Menno van Lookeren Campagne; Peter Vandenabeele; Wim Vandenberghe; Ilse Vanhorebeek; Isabel Varela-Nieto; M Helena Vasconcelos; Radovan Vasko; Demetrios G Vavvas; Ignacio Vega-Naredo; Guillermo Velasco; Athanassios D Velentzas; Panagiotis D Velentzas; Tibor Vellai; Edo Vellenga; Mikkel Holm Vendelbo; Kartik Venkatachalam; Natascia Ventura; Salvador Ventura; Patrícia St Veras; Mireille Verdier; Beata G Vertessy; Andrea Viale; Michel Vidal; Helena L A Vieira; Richard D Vierstra; Nadarajah Vigneswaran; Neeraj Vij; Miquel Vila; Margarita Villar; Victor H Villar; Joan Villarroya; Cécile Vindis; Giampietro Viola; Maria Teresa Viscomi; Giovanni Vitale; Dan T Vogl; Olga V Voitsekhovskaja; Clarissa von Haefen; Karin von Schwarzenberg; Daniel E Voth; Valérie Vouret-Craviari; Kristina Vuori; Jatin M Vyas; Christian Waeber; Cheryl Lyn Walker; Mark J Walker; Jochen Walter; Lei Wan; Xiangbo Wan; Bo Wang; Caihong Wang; Chao-Yung Wang; Chengshu Wang; Chenran Wang; Chuangui Wang; Dong Wang; Fen Wang; Fuxin Wang; Guanghui Wang; Hai-Jie Wang; Haichao Wang; Hong-Gang Wang; Hongmin Wang; Horng-Dar Wang; Jing Wang; Junjun Wang; Mei Wang; Mei-Qing Wang; Pei-Yu Wang; Peng Wang; Richard C Wang; Shuo Wang; Ting-Fang Wang; Xian Wang; Xiao-Jia Wang; Xiao-Wei Wang; Xin Wang; Xuejun Wang; Yan Wang; Yanming Wang; Ying Wang; Ying-Jan Wang; Yipeng Wang; Yu Wang; Yu Tian Wang; Yuqing Wang; Zhi-Nong Wang; Pablo Wappner; Carl Ward; Diane McVey Ward; Gary Warnes; Hirotaka Watada; Yoshihisa Watanabe; Kei Watase; Timothy E Weaver; Colin D Weekes; Jiwu Wei; Thomas Weide; Conrad C Weihl; Günther Weindl; Simone Nardin Weis; Longping Wen; Xin Wen; Yunfei Wen; Benedikt Westermann; Cornelia M Weyand; Anthony R White; Eileen White; J Lindsay Whitton; Alexander J Whitworth; Joëlle Wiels; Franziska Wild; Manon E Wildenberg; Tom Wileman; Deepti Srinivas Wilkinson; Simon Wilkinson; Dieter Willbold; Chris Williams; Katherine Williams; Peter R Williamson; Konstanze F Winklhofer; Steven S Witkin; Stephanie E Wohlgemuth; Thomas Wollert; Ernst J Wolvetang; Esther Wong; G William Wong; Richard W Wong; Vincent Kam Wai Wong; Elizabeth A Woodcock; Karen L Wright; Chunlai Wu; Defeng Wu; Gen Sheng Wu; Jian Wu; Junfang Wu; Mian Wu; Min Wu; Shengzhou Wu; William Kk Wu; Yaohua Wu; Zhenlong Wu; Cristina Pr Xavier; Ramnik J Xavier; Gui-Xian Xia; Tian Xia; Weiliang Xia; Yong Xia; Hengyi Xiao; Jian Xiao; Shi Xiao; Wuhan Xiao; Chuan-Ming Xie; Zhiping Xie; Zhonglin Xie; Maria Xilouri; Yuyan Xiong; Chuanshan Xu; Congfeng Xu; Feng Xu; Haoxing Xu; Hongwei Xu; Jian Xu; Jianzhen Xu; Jinxian Xu; Liang Xu; Xiaolei Xu; Yangqing Xu; Ye Xu; Zhi-Xiang Xu; Ziheng Xu; Yu Xue; Takahiro Yamada; Ai Yamamoto; Koji Yamanaka; Shunhei Yamashina; Shigeko Yamashiro; Bing Yan; Bo Yan; Xianghua Yan; Zhen Yan; Yasuo Yanagi; Dun-Sheng Yang; Jin-Ming Yang; Liu Yang; Minghua Yang; Pei-Ming Yang; Peixin Yang; Qian Yang; Wannian Yang; Wei Yuan Yang; Xuesong Yang; Yi Yang; Ying Yang; Zhifen Yang; Zhihong Yang; Meng-Chao Yao; Pamela J Yao; Xiaofeng Yao; Zhenyu Yao; Zhiyuan Yao; Linda S Yasui; Mingxiang Ye; Barry Yedvobnick; Behzad Yeganeh; Elizabeth S Yeh; Patricia L Yeyati; Fan Yi; Long Yi; Xiao-Ming Yin; Calvin K Yip; Yeong-Min Yoo; Young Hyun Yoo; Seung-Yong Yoon; Ken-Ichi Yoshida; Tamotsu Yoshimori; Ken H Young; Huixin Yu; Jane J Yu; Jin-Tai Yu; Jun Yu; Li Yu; W Haung Yu; Xiao-Fang Yu; Zhengping Yu; Junying Yuan; Zhi-Min Yuan; Beatrice Yjt Yue; Jianbo Yue; Zhenyu Yue; David N Zacks; Eldad Zacksenhaus; Nadia Zaffaroni; Tania Zaglia; Zahra Zakeri; Vincent Zecchini; Jinsheng Zeng; Min Zeng; Qi Zeng; Antonis S Zervos; Donna D Zhang; Fan Zhang; Guo Zhang; Guo-Chang Zhang; Hao Zhang; Hong Zhang; Hong Zhang; Hongbing Zhang; Jian Zhang; Jian Zhang; Jiangwei Zhang; Jianhua Zhang; Jing-Pu Zhang; Li Zhang; Lin Zhang; Lin Zhang; Long Zhang; Ming-Yong Zhang; Xiangnan Zhang; Xu Dong Zhang; Yan Zhang; Yang Zhang; Yanjin Zhang; Yingmei Zhang; Yunjiao Zhang; Mei Zhao; Wei-Li Zhao; Xiaonan Zhao; Yan G Zhao; Ying Zhao; Yongchao Zhao; Yu-Xia Zhao; Zhendong Zhao; Zhizhuang J Zhao; Dexian Zheng; Xi-Long Zheng; Xiaoxiang Zheng; Boris Zhivotovsky; Qing Zhong; Guang-Zhou Zhou; Guofei Zhou; Huiping Zhou; Shu-Feng Zhou; Xu-Jie Zhou; Hongxin Zhu; Hua Zhu; Wei-Guo Zhu; Wenhua Zhu; Xiao-Feng Zhu; Yuhua Zhu; Shi-Mei Zhuang; Xiaohong Zhuang; Elio Ziparo; Christos E Zois; Teresa Zoladek; Wei-Xing Zong; Antonio Zorzano; Susu M Zughaier Journal: Autophagy Date: 2016 Impact factor: 16.016
Authors: Monica Benvenuto; Sara Ciuffa; Chiara Focaccetti; Diego Sbardella; Sara Fazi; Manuel Scimeca; Grazia Raffaella Tundo; Giovanni Barillari; Maria Segni; Elena Bonanno; Vittorio Manzari; Andrea Modesti; Laura Masuelli; Massimo Coletta; Roberto Bei Journal: Sci Rep Date: 2021-09-24 Impact factor: 4.379
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