Figure 2
Figure 2. Caspase-4 acts upstream of caspase-8 and -3 processing during spontaneous B-CLL cell apoptosis. (A) Time-course analysis of caspase-8 and -3 proteolytic processing during spontaneous B-CLL cell apoptosis. Western blot analysis was performed in 25 μg whole-cell lysates extracted from freshly isolated B-CLL cells and B-CLL cells cultured in complete medium for the indicated times (n = 12). Protein loading was assessed by reprobing the blots with an anti–β-actin mAb. Vertical lines inserted in caspase-3 blot indicate a repositioned gel lane. The data shown for patients 1 and 2 are representative of 12 patients. (B) Effect of pharmacologic inhibition of caspase-8 and -3 on spontaneous B-CLL cell apoptosis. Apoptosis was evaluated by flow cytometric analysis of hypodiploid nuclei in B-CLL cells cultured for 24 hours in complete medium with 50μM of the caspase-8 inhibitor z-IETD-fmk, 50μM of the caspase-3 inhibitor z-DEVD-fmk, or 0.005% DMSO as control (n = 7). Results are the mean ± SD of all 7 patients examined. **P < .01 (each caspase inhibitor vs DMSO) according to Student t test. (C) Effect of pharmacologic caspase-4 inhibition on caspase-8 and -3 proteolytic processing and PARP degradation. Caspase-8 and -3 processing and PARP degradation were analyzed by Western blot in 25 μg whole-cell lysates extracted from B-CLL cells cultured for 24 hours in complete medium with 50μM of the caspase-4 inhibitor z-LEVD-fmk or 0.005% DMSO as control (n = 6). Protein loading was assessed by reprobing the blots with an anti–β-actin mAb. The data shown for patients 1 and 2 are representative of 6 patients. (D) Effect of caspase-4 down-regulation on caspase-8 and -3 proteolytic processing. Freshly isolated B-CLL cells were transfected with 0.5μM control nontargeting (siCtrl) or caspase-4 siRNA (siCasp-4) as described in “siRNA nucleofection” and then cultured in complete medium for 72 hours (n = 6). Caspase-4, -8, and -3 processing was analyzed by Western blot in 25 μg whole-cell lysates, and protein loading was assessed by reprobing the blots with an anti–β-actin mAb. In caspase-4 blots, separated panels are shown because long X-ray film exposure was necessary to detect the 20-kDa subunit. The asterisk in caspase-4 blots indicates bands derived from unknown cleavage. Data shown for patients 1 and 2 are representative of 6 patients.

Caspase-4 acts upstream of caspase-8 and -3 processing during spontaneous B-CLL cell apoptosis. (A) Time-course analysis of caspase-8 and -3 proteolytic processing during spontaneous B-CLL cell apoptosis. Western blot analysis was performed in 25 μg whole-cell lysates extracted from freshly isolated B-CLL cells and B-CLL cells cultured in complete medium for the indicated times (n = 12). Protein loading was assessed by reprobing the blots with an anti–β-actin mAb. Vertical lines inserted in caspase-3 blot indicate a repositioned gel lane. The data shown for patients 1 and 2 are representative of 12 patients. (B) Effect of pharmacologic inhibition of caspase-8 and -3 on spontaneous B-CLL cell apoptosis. Apoptosis was evaluated by flow cytometric analysis of hypodiploid nuclei in B-CLL cells cultured for 24 hours in complete medium with 50μM of the caspase-8 inhibitor z-IETD-fmk, 50μM of the caspase-3 inhibitor z-DEVD-fmk, or 0.005% DMSO as control (n = 7). Results are the mean ± SD of all 7 patients examined. **P < .01 (each caspase inhibitor vs DMSO) according to Student t test. (C) Effect of pharmacologic caspase-4 inhibition on caspase-8 and -3 proteolytic processing and PARP degradation. Caspase-8 and -3 processing and PARP degradation were analyzed by Western blot in 25 μg whole-cell lysates extracted from B-CLL cells cultured for 24 hours in complete medium with 50μM of the caspase-4 inhibitor z-LEVD-fmk or 0.005% DMSO as control (n = 6). Protein loading was assessed by reprobing the blots with an anti–β-actin mAb. The data shown for patients 1 and 2 are representative of 6 patients. (D) Effect of caspase-4 down-regulation on caspase-8 and -3 proteolytic processing. Freshly isolated B-CLL cells were transfected with 0.5μM control nontargeting (siCtrl) or caspase-4 siRNA (siCasp-4) as described in “siRNA nucleofection” and then cultured in complete medium for 72 hours (n = 6). Caspase-4, -8, and -3 processing was analyzed by Western blot in 25 μg whole-cell lysates, and protein loading was assessed by reprobing the blots with an anti–β-actin mAb. In caspase-4 blots, separated panels are shown because long X-ray film exposure was necessary to detect the 20-kDa subunit. The asterisk in caspase-4 blots indicates bands derived from unknown cleavage. Data shown for patients 1 and 2 are representative of 6 patients.

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