Figure 6.

SIRT5 promoted autophagy in HCG cells via AMPK–mTOR-regulated autophagy, SIRT5 is degraded during etoposide-induced apoptosis, and calpains and caspases are responsible for the cleavage of SIRT5. (a) Western blot analysis of p-AMPK, t-AMPK, p-mTOR, and t-mTOR in vector-transfected or SIRT5-OE HCG27 cells induced by 100 nM rapamycin. (b–c) Relative protein levels were determined after normalization to t-AMPK or t-mTOR. (d) AGS cells were treated with 1 mg/mL etoposide for the indicated times. SIRT5, Beclin 1, cleaved PARP, and ATG5 expression was detected via western blotting at various time points. (e) AGS cells were treated with 1 mg/mL etoposide for the indicated times, and pro-caspase-3 and cleaved caspase-3 expression was analyzed via western blotting. (f) Normalized quantitation of the optical density of SIRT5, Beclin 1, cleaved PARP, and ATG5 in AGS cells treated with etoposide for the indicated times. (g) Normalized quantitation of the optical density of pro-caspase-3 and cleaved caspase-3 in AGS cells treated with etoposide for the indicated times. (h) AGS cells treated with 1 mg/mL etoposide for 6 hours in the presence or absence of 20 mM calpain inhibitor (CL) and/or 10 mM caspase inhibitor (z-VAD-fmk). SIRT5 expression was evaluated via western blotting. All data represent the average and SD of three or six independent experiments (*P < 0.05).

SIRT5, sirtuin 5; AMPK, AMP-activated protein kinase, mTOR, mammalian target of rapamycin; p-AMPK, phosphorylated AMPK; t-AMPK, total AMPK; p-mTOR, phosphorylated mTOR; t-mTOR; total mTOR; SIRT5-OE, sirtuin 5-overexpressing lentivirus; Vector, control lentivirus; PARP, poly(ADP-ribose) polymerase; ATG5, autophagy-related 5.