DZNep-mediated sensitization to TRAIL correlates with enhanced activation of caspase signaling and involvement of the mitochondrial apoptosis pathway.

<p>(<b>A</b>) Analysis of caspase-8, caspase-3, and PARP processing of cells pre-treated with DZNep (1 μM, 24 h) followed by TRAIL (10 or 20 ng/ml) stimulation for 6 or 16 h. Cells were incubated with pan-caspase inhibitor ZVAD-fmk (20 μM) for 3 h prior to DZNep treatment. Protein transfer was confirmed by ponceau S staining, and equal loading was confirmed by GAPDH. (<b>B</b>) Apoptosis induction determined by subG1 analysis of cells pre-treated with DZNep (1 μM, 24 h) and treated with TRAIL (10 ng/ml, 16 h); ZVAD-fmk (20 μM) was given for 3 h prior to DZNep. (<b>C</b>) Activation of the mitochondrial apoptosis pathway was determined by TMRM staining for mitochondrial membrane potential (Δψ_m) in Z-138 cells treated with DZNep (0.2, 1, 3, or 5 μM) alone for 40 h. (<b>D, E</b>) Analysis of Δψ_m of cells (Z-138, BJAB, and SU-DHL-9) treated with DZNep (1 μM, 24 h) followed by TRAIL (10 ng/ml for 1–5 h or 20 ng/ml for 16 h). Shown are percentages of cells with decreased Δψ_m.