Proteasome inhibitors induce apoptosis in a caspase-dependent fashion. (A) Jurkat cells were exposed for 24 hours to the indicated concentrations of proteasome inhibitors (♦, epoxomicin; ▪, lactacystin; •, MG132; ▴, ZL3VS). Thereafter, cells were harvested, and dead cells were quantified by flow cytometry after staining with propidium iodide. Means of duplicates are shown. (B) Jurkat cells were preincubated for 1 hour in the presence (□) or absence (▪) of zVAD-fmk (100 μM) and thereafter were exposed to epoxomicin (0.4 μM), MG132 (0.5 μM), lactacystin (5 μM), ZL3VS (10 μM), or regular medium for 24 hours. Subsequently, cell death was quantified by flow cytometric analysis of propidium iodide-stained cells. Mean values of duplicates are presented. (C) Jurkat cells were preincubated for 1 hour in the presence or absence of zVAD-fmk and thereafter were cultured in the presence or absence of epoxomicin (0.4 μM), MG132 (1 μM), lactacystin (5 μM), or ZL3VS (10 μM) for 8 hours. Subsequently, lysates were prepared and cellular proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE). Caspase activation was detected by cleavage of caspase-3, caspase-8, and caspase-9 using immunoblot analysis. (D) Following a 1-hour preincubation with or without zVAD-fmk, Jurkat cells were stimulated with regular medium, epoxomicin (0.4 μM), etoposide (25 μg/mL), or TRAIL (100 ng/mL) for the indicated time amounts. Cleavage of caspase-3, caspase-8, caspase-9, and PARP was detected using immunoblot analysis.
