Figure 1
Involvement of ER stress-associated signaling in spontaneous B-CLL cell apoptosis. (A,F,G) Time-course analysis of caspase-4 proteolytic processing, CHOP/GADD153 and BiP/GRP78 expression, and JNK1/2 phosphorylation during spontaneous B-CLL cell apoptosis. Western blot analysis was performed in whole-cell lysates (25 μg for caspase-4, BiP/GRP78, and JNK1/2, and 40 μg for CHOP/GADD153) 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. 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. The density of the bands corresponding to CHOP/GADD153 and BiP/GRP78 was evaluated by densitometric analysis. Densitometry units (U) were calculated relative to β-actin. The data shown for patients 1 and 2 are representative of 12 patients. (B) Effect of pharmacologic caspase-4 inhibition 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 or without 50μM of the caspase-4 inhibitor z-LEVD-fmk, 50μM of the pan-caspase inhibitor z-VAD-fmk, or 0.005% DMSO as control (n = 7). Results are presented as the mean ± SD of all 7 patients examined. **P < .01 (each caspase inhibitor vs DMSO) according to Student t test. (C-E) Effect of caspase-4 down-regulation on spontaneous B-CLL cell apoptosis. 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 = 7). Caspase-4 expression (C) was analyzed by Western blot as described in panel A. Apoptosis was evaluated by flow cytometric analysis of annexin V/PI-stained cells (D) and hypodiploid nuclei (E). (D) Results are presented as the percentage of viable (annexin V−/PI−), early apoptotic (annexin V+/PI−), late apoptotic (annexin V+/PI+), and necrotic cells (annexin V−/PI+). (E) Percentage of hypodiploid nuclei. (C-E) Results shown for patients 1, 2, and 3 are representative of 7 patients.

Involvement of ER stress-associated signaling in spontaneous B-CLL cell apoptosis. (A,F,G) Time-course analysis of caspase-4 proteolytic processing, CHOP/GADD153 and BiP/GRP78 expression, and JNK1/2 phosphorylation during spontaneous B-CLL cell apoptosis. Western blot analysis was performed in whole-cell lysates (25 μg for caspase-4, BiP/GRP78, and JNK1/2, and 40 μg for CHOP/GADD153) 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. 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. The density of the bands corresponding to CHOP/GADD153 and BiP/GRP78 was evaluated by densitometric analysis. Densitometry units (U) were calculated relative to β-actin. The data shown for patients 1 and 2 are representative of 12 patients. (B) Effect of pharmacologic caspase-4 inhibition 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 or without 50μM of the caspase-4 inhibitor z-LEVD-fmk, 50μM of the pan-caspase inhibitor z-VAD-fmk, or 0.005% DMSO as control (n = 7). Results are presented as the mean ± SD of all 7 patients examined. **P < .01 (each caspase inhibitor vs DMSO) according to Student t test. (C-E) Effect of caspase-4 down-regulation on spontaneous B-CLL cell apoptosis. 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 = 7). Caspase-4 expression (C) was analyzed by Western blot as described in panel A. Apoptosis was evaluated by flow cytometric analysis of annexin V/PI-stained cells (D) and hypodiploid nuclei (E). (D) Results are presented as the percentage of viable (annexin V−/PI−), early apoptotic (annexin V+/PI−), late apoptotic (annexin V+/PI+), and necrotic cells (annexin V−/PI+). (E) Percentage of hypodiploid nuclei. (C-E) Results shown for patients 1, 2, and 3 are representative of 7 patients.

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