Impact of Z-VAD, a pan-caspase inhibitor on cell death. Impact of Z-VAD on cell-death–associated proteins (A) and cell death (B-E). (A) CLL lymphocytes from patient 29 were cultured in suspension or on stroma. These were pretreated with or without Z-VAD and then either untreated or treated with bendamustine, fludarabine, or their combination for 48 hours, harvested, and lysed. Immunoblot analysis of the total and phosphoprotein levels of p53 and total PUMA, Bcl-2, Mcl-1, and cleaved and uncleaved PARP was performed. GAPDH was used as a protein loading control. (B-E) CLL cells from patients (n = 3) cultured in suspension (B-C) or on stroma (D-E) were either untreated (−) or pretreated (+) with Z-VAD followed by a 24- or 48-hour treatment with bendamustine and fludarabine together and assayed for DiOC6 negativity (B,D) or annexin V/PI positivity (C,E). Data are presented as mean with SD from samples from 3 patients, and results are corrected to a time-matched control (untreated sample) for each patient sample. B, bendamustine; C, control (untreated); F, fludarabine.