Figure 4.

Suppression of autophagy enhances proteasome inhibitor-induced cell death in cancer cells. A: Bax-positive and Bax-deficient HCT 116 cells were treated with MG132 in the presence (closed column) or absence (open column) of 3-MA (10 mmol/L) for 24 hours and then stained with Hoechst 33328. Cells with apoptotic nuclear features were defined as apoptotic cells. *P < 0.01 and ^$P < 0.05 by Z-test between groups without 3-MA and groups with 3-MA for each MG132 dosage and cell line. B: Bax-deficient HCT 116 and DU145 cells were transfected with indicated siRNA against specific Atg genes (target) or a negative control siRNA (Ctrl) for 48 hours followed by immunoblot with different antibodies. Atg5 was detected as the complex with Atg12. C: Bax-deficient HCT 116 (white column) and DU145 (gray column) cells were transfected with 120 nmol/L siRNA against Atg8/LC3B or a negative control siRNA for 36 hours before being treated with MG132. The number of AVs per 100 μm² of cytoplasm area (means ± SD) was then quantified. * and ^#P < 0.01 by one-way analysis of variance for both cell lines (*MG132 plus negative siRNA versus the control; ^#MG132 plus siRNA-LC3B versus MG132 plus negative siRNA). D and E: Bax-deficient HCT 116 cells were transfected with specific siRNA or a negative control (Neg) as indicated and treated with MG132 (0.25 μmol/L) for 16 hours. Apoptotic cells (D) were determined as in A. *P < 0.01 by Z-test (MG132 plus specific siRNA versus MG132 plus negative siRNA). Effector caspase activities (E) were measured using DEVD-AFC as the substrate, and the results were expressed as the fold increase over the nontreated control group.