Fig. 8. Rapamycin-induced autophagy ameliorates GSH depletion-induced premature cell senescence.

a–c ARPE-19 cells were coincubated with Cys₂ starvation milieu, 1000 µM BSO, and 10 µM erastin supplied with autophagic inducer rapamycin (100 nM), autophagic inhibitor 3-MA (10 mM) and lysosomal inhibitor Baf-A1 (75 nM) for 24 h; representative images of RPE cells displaying LC3 puncta were immediately visualized by confocal microscopy. Mean number of autophagosomes represented by yellow puncta in merged images and autolysosomes represented by red puncta in merged images per cell. Scale bar, 10 µm. ** represent p < 0.01. d Images of senescence β-galactosidase staining. Cells were treated with Cys₂ starvation, 1000 µM BSO, and 10 µM erastin with or without 100 nM rapamycin and 10 mM 3-MA for 24 h, respectively. Scale bars: 50 μm. e Downregulation of p16 after coincubation with autophagic inducer rapamycin, while upregulation of p16 after coincubation with autophagic inhibitor 3-MA and lysosomal inhibitor Baf-A1 were detected by western blotting