Caspase-8 is involved in Bap31 cleavage and mitochondrial cytochrome c release during spontaneous B-CLL cell apoptosis. (A,C) Time-course analysis of Bap31 cleavage and mitochondrial cytochrome c release during spontaneous B-CLL cell apoptosis. Freshly isolated B-CLL cells were cultured for the indicated times in complete medium (n = 12). Bap31 cleavage (A) was analyzed by Western blot in 20 μg whole-cell lysates, and protein loading was assessed by reprobing the blots with an anti–β-actin mAb. The density of the bands corresponding to Bap20 fragment was evaluated by densitometric analysis. Densitometry units (U) were calculated relative to β-actin. The release of cytochrome c from the mitochondria into the cytosol (C) was analyzed by Western blot in 20 μg of cytosol and mitochondria-enriched fraction (MF) extracts. The blots were reprobed with an anti-COX IV mAb to control the purity of cytosolic fraction and the loading of MF, and with an anti–β-actin mAb as a loading control for cytosolic extracts. (A,C) Data shown for patients 1 and 2 are representative of 12 patients. (B,D) Effect of pharmacologic caspase-8 inhibition on Bap31 cleavage and mitochondrial cytochrome c release. B-CLL cells were cultured for 24 hours in complete medium with 50μM of the caspase-8 inhibitor z-IETD-fmk or 0.005% DMSO as control (n = 7). Bap31 cleavage (B) was analyzed as described in panel A. Cytochrome c levels (D) were analyzed by Western blot in cytosolic fraction (20 μg). The blots were reprobed with an anti-COX IV mAb and an anti–β-actin mAb to control the purity and the loading of cytosolic fraction, respectively. (B,D) Data shown for patients 1 and 2 are representative of 7 patients.