The caspase inhibitor z-VAD-fmk does not prevent OSU03012-mediated cell death. (A) The caspase inhibitor z-VAD-fmk does not prevent OSU03012-mediated cell death. After exposure to 10 μM OSU03012 or 5 μM fludarabine for 24 hours, PBMCs from patients with CLL were resuspended in binding buffer containing annexin V–FITC and propidium iodide and assessed by flow cytometry. Some cells were incubated with 100 μM z-VAD-fmk, the cell-permeable pan-caspase inhibitor. Data are representative of 5 patients. (B) OSU03012 induces apoptosis independent of caspase activation. CLL cells were incubated in media, 100μM z-VAD-fmk, 10 μM OSU03012, or 100 μM z-VAD-fmk and 10 μM OSU03012 followed by assessment of viability by annexin V/PI staining 24 hours later. The percentage of viable cells was not influenced with z-VAD-fmk. Data represent mean of 5 patients. Error bars are standard deviation. (C) Protein lysates from cells treated with media (M), 100 μM z-VAD-fmk (Z), 10 μM OSU03012 (12), or a combination were probed for PARP by immunoblot. Positive control is lysate from irradiated Jurkat cells. Blot is representative of 4 experiments. (D) Protein lysates from cells treated with media (M), 50 μM z-VAD-fmk (Z), 10 μM OSU03012 (12), or a combination were probed for caspase-3 by immunoblot. Positive control is lysate from irradiated Jurkat cells. Blot is representative of 4 experiments.