Figure 7.

SIRT1 activation is a key determinant of the PARP2-mediated inhibition of autophagy. (A) scPARP2 and shPARP2 C2C12 cells were treated with 50 µM resveratrol or 25 µM EX-527 for 24 h (n = 3). LC3 expression was assessed by confocal microscopy. Alexa Fluor 488-linked LC3 specific antibody was used and the nuclei were visualized using DAPI and vesicles were counted. Representative images are presented on the figure. (B,C) SIRT1 was transiently silenced in scPARP2 and shPARP2 C2C12 cells using siRNA targeting SIRT1 (n = 3). Cells were transfected with siRNA for 48 h, then LC3 expression was assessed in confocal microscopy experiments and the protein level of SIRT1 was detected by Western blotting. Alexa Fluor 488-inked LC3 specific antibody was used and the nuclei were visualized using DAPI. Representative images are presented in the figure. Cells were scored as described in Materials and Methods. # and ### represent statistically significant differences between the control and treated cells at p < 0.05 and p < 0.001, respectively. *** represent statistically significant differences between the scPARP2 and shPARP2 cells at p < 0.001. For the determination of statistical significance, ANOVA test was used followed by Tukey’s post hoc test.