Figure 6

Autophagy protection of MG132-treated Cas-deficient cells. (A) Time course of MG132-induced autophagy and apoptosis in Cas-/- (EV) and Cas-FL MEFs. Cas-/- (EV) and Cas-FL MEFs were treated with 1 μM MG132 as indicated, and LC3-II expression and PARP cleavage were examined by immunoblot. (B) Effect of autophagy on MG132-induced apoptosis. MG132-treated Cas-/- (EV) or Cas-FL cells were assayed by immunoblot to detect the PARP cleavage in the presence of autophagy inhibitors 3-MA (10 mM) or CHQ (20 μM). (C) Apoptosis ELISA (left panel) and MTS assay (right panel) were used to examine the effects of autophagy inhibition on MG132-induced cell death in Cas-/- (EV) and Cas-FL MEFs. (D) Cas-/-(EV) and Cas-FL MEFs stably transfected with ATG5 shRNAs or control shRNA were treated with 1 μM MG132, and the cleavage of PARP and caspase-3 was examined by immunoblot (right panel). ATG5 expression in the various cell lines as measured by immunoblot is shown in the left panels. The effect of shRNA-mediated knockdown of ATG5 on LC3-II formation is shown in the middle panels, as detected by immunoblot. (E) ATG5 shRNA-transfected MEFs were treated with 1 μM MG132, and cell death was examined by the apoptosis ELISA assay (left panel) or by MTS assay (right panel).