Fig 2: eIF2α is involved in the regulation of eEF2 phosphorylation in response to ER stress. eEF2K (A) and eEF2 (B) were immunoprecipitated from lysates of H9c2 cells treated or not with 10 μg/mL tunicamycin (TN), and the presence of eEF2K or eEF2 and eIF2α were analyzed by Western blot (n = 3). (C,D) H9c2 cells were transfected with control or eIF2α siRNA for 24 h, treated with tunicamycin (TN) for 1h30, and the level of eIF2α or phospho-eEF2 was analyzed by Western blot (RD: Relative density). The ratio of p-eEF2 vs. total eEF2 is presented in the bar graph. Error bars, S.E.M. (n = 4). ** P < 0.01 versus CTL siRNA+TN.

Fig 3: SIRT1 activates eEF2K/eEF2-dependent autophagy in response to ER stress. (A) H9c2 cells were treated with 10 µM A484954 for 4 h (eEF2K inhibitor) and the level of phospho-eEF2 (p-eEF2) was analyzed by Western blot (n = 7). The ratio of p-eEF2 vs. total eEF2 is presented. (B) H9c2 cells were left untreated or treated with 10 µg/mL tunicamycin (TN) for 48 h ± 10 µM A484954, and cell viability was determined by Fluorescein Diacetate (FDA) assay. Results presented in the bar graph are expressed as mean ± S.E.M. of percentages of dead cells (FDA negative cells, n = 11). (C) Autophagy was monitored by flow cytometry after Cyto-ID® staining of H9c2 cells untreated or treated with 10 µg/mL tunicamycin (TN) for 24 h ± 10 µM A484954 (n = 11). (D) Cells were left untreated or treated with 10 µg/mL tunicamycin (TN) for 24 h ± 10 µM A484954 and the levels of LC3-II were analyzed by Western blot. Actin was used as a loading control. The relative mean expression level of LC3-II is presented in the bar graph. Error bars, S.E.M. (n = 5). (E,F,I) H9c2 cells were transfected with control, eEF2K or eEF2 siRNA, and eEF2K (E) or eEF2 (I) expression and eEF2 phosphorylation (F,I) were assessed after 24 h by Western blot. (G,J) H9c2 cells were transfected with control, eEF2K (G), or eEF2 (J) siRNA for 24 h, then treated for 48 h with tunicamycin (TN) and cell death (FDA negative cells) was measured by flow cytometry. Data in the bar graphs represent mean ± S.E.M. (n = 3). (H,K) H9c2 cells were transfected with control, eEF2K (H) or eEF2 (K) siRNA for 24 h, then treated for 24 h with tunicamycin (TN) and autophagy (MFI of Cyto-ID®) was measured by flow cytometry. Data in the bar graphs represent mean ± S.E.M. (n = 3). (L) phospho-eEF2 expression level was measured by Western blot in H9c2 cells after 4 h of 10 µg/mL tunicamycin (TN) ± 50µM EX527 (SIRT1 inhibitor) treatment. The ratio of p-eEF2 vs. total eEF2 is presented in the bar graph. Error bars, S.E.M. (n = 9). * P < 0.05, ** P < 0.01, *** P < 0.005 versus control. # P < 0.05, ### P < 0.005 versus TN.