Effect of BiP/GRP78 down-regulation on spontaneous B-CLL cell apoptosis. (A-C) Freshly isolated B-CLL cells were transfected with 2.0μM control nontargeting (siCtrl) or BiP/GRP78 siRNA (siBiP) as described in “siRNA nucleofection” and then cultured in complete medium for 72 hours (n = 7). (A) BiP/GRP78 expression 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. For each sample, blot lanes corresponding to siCtrl and siBiP were subjected to densitometric analysis and normalized to β-actin levels, and values under siBiP lane of each sample represent the percentage inhibition of BiP/GRP78 expression induced by siBiP compared with siCtrl. (B-C) Apoptosis was evaluated by flow cytometric analysis of hypodiploid nuclei (B) and annexin V/PI-stained cells (C). (A-B) Results from all 7 patients examined are shown. (C) Results shown for patients 13, 15, and 16 are the percentage of viable (annexin V⁻/PI⁻), early apoptotic (annexin V⁺/PI⁻), late apoptotic (annexin V⁺/PI⁺), and necrotic cells (annexin V⁻/PI⁺).