Proteasome inhibitor-induced cytochrome c/SMAC/Diablo release and mitochondrial depolarization is inhibited by Bcl-2. (A) Jurkat cells were preincubated for 1 hour in the presence or absence of zVAD-fmk and thereafter were cultured in the presence of MG132 (0.5 μM), etoposide (25 μg/mL), or TRAIL (100 ng/mL) for the indicated time amounts. Subsequently, the cytosolic fraction was isolated by resuspending the cells in a digitonin-containing lysis buffer. Cytochrome c levels (cyt c) in the cytosol were detected by immunoblotting. A nonspecific crossreactive immune band (ns) was used as an equal protein loading control. (B) Bcl-2-overexpressing Jurkat cells and respective vector control cells were preincubated for 1 hour with or without zVAD-fmk and subsequently stimulated with 0.4 μM epoxomicin for the indicated time amounts. Following isolation of the cytosolic fraction, cytochrome c and SMAC/Diablo relocalization was detected by immunoblotting. (C) Jurkat cells were incubated in the presence of epoxomicin (0.4 μM; ▴), etoposide (25 μg/mL; ▪), or TRAIL (100 ng/mL; ♦). Cells were harvested at the indicated amounts of time and stained with TMRE, and

cells were enumerated by flow cytometry. (D) Bcl-2 and vector control Jurkat cells were stimulated (bold line) or not (thin line) with epoxomicin (0.4 μM), MG132 (0.5 μM), or etoposide (25 μg/mL) for 18 hours. Subsequently, cells were harvested, stained with TMRE, and analyzed by flow cytometry.