Silencing of Mcl-1, Bcl-xL, and XIAP by RNA interference in primary CLL B cells and in the MEC1 cell line. (A) Leukemic B cells were nucleofected with Mcl-1, Bcl-xL, XIAP, or control siRNAs and analyzed by immunoblotting after 24 and 48 hours. Experiments with 1 representative CLL B-cell sample (G147) and with the CLL cell line MEC1 are shown. The percentage of viable cells was determined for each sample by Annexin V/PI staining and is indicated at the bottom of the panel. Induction of apoptosis was also evidenced by increased cleavage of the caspase 3 substrate PARP in CLL cells transfected with the Mcl-1–specific mRNA. (B) CLL cells were nucleofected with control or Mcl-1–specific siRNA and cultured with or without the pancaspase inhibitor Z-VAD-fmk (100 μM). Expression of Mcl-1 and XIAP was evaluated by immunoblotting 24 hours after the nucleofection. The levels of Mcl-1 and XIAP were normalized to the levels of Bcl-2, which served as a loading control, and are presented as fold change relative to the control nucleofection. The percentage of viable cells is indicated at the bottom of the panel. One representative experiment out of 3 with similar results is shown. (C) MEC1 cells were cotransfected with the Mcl-1 and XIAP siRNAs to evaluate the effect of simultaneous inhibition of both proteins. Apoptosis induction and expression of Mcl-1 and XIAP was evaluated as in panel B.