Figure 5.

The pan caspase inhibitor V-ZAD-fmk inhibits coibamide-induced cytotoxicity in MEFs. (A) Cytoprotective effect of V-ZAD-fmk on both wild-type and ATG5-null mouse embryonic fibroblasts (MEFs) treated with coibamide A. Cells were exposed to increasing concentrations of coibamide A (0.3 nM to 3 µM), with or without V-ZAD-fmk (50 µM), and the viability was determined with a WST-8 proliferation/cytotoxicity assay at 24 h. The viability of vehicle-treated cells was defined as 100%. Data points show mean viability ± SE (n = 3 wells per treatment) from a representative comparison that was repeated in three independent experiments. (B) Expression of endogenous biomarkers of autophagy and caspase-dependent apoptosis in wild-type MEFs at 24 h. Immunoblot analysis of: poly [ADP-ribose] polymerase 1 (PARP-1), cleaved caspase-3 and LC3-I/II relative to alpha-tubulin and acetyl-CoA carboxylase (ACC), in cells treated with, or without (0), vehicle (0.1% DMSO) or coibamide A (3–30 nM) for 24 h. Whole cell lysates were probed with appropriate primary antibodies as indicated. Cleavage product of PARP-1 is denoted by an arrow. Each series of blots is representative of patterns that were observed in at least three independent experiments.