Effect of depsipeptide on caspase 3 activation and PARP cleavage. (A) Depsipeptide treatment results in activation of caspase 3 as assessed by flow cytometry with the use of a PE-directed antibody specific for the active cleavage product of caspase 3. Depsipeptide in human CLL cells activates caspase 3. CLL cells were treated with media or depsipeptide (0.038 and 0.38 μM) for 4 hours and subsequently incubated in media for 20 hours. Cells were washed, permeated, and stained with a PE-directed antibody specific for the active cleavage product of caspase 3. (B) Depsipeptide treatment results in cleavage of PARP. Depsipeptide-mediated apoptosis promotes processing of PARP. To determine if depsipeptide treatment caused alteration of a caspase 3 substrate, we examined both the unprocessed and the processed forms of PARP in fresh human CLL cells at 24 hours following a 4-hour incubation of CLL cells with media, 0.038 μM depsipeptide, or 0.38 μM depsipeptide. Protein lysates were prepared, and 50 μg protein per lane was separated on a 14% SDS-PAGE gel. Loading equivalence was confirmed by blotting with an antibody for constitutively expressed protein GAPDH. PARP and its cleaved product were detected by means of an anti-PARP polyclonal antibody.