Resistance of BCR-stimulated cells to ABT-199 is associated with Mcl-1 upregulation. (A) CLL cells were cultured for 24 hours with or without ABT-199 or ABT-199 + imm-aIgM. ABT-199 (2 nM) was added 3 hours after imm-aIgM. Cellular extracts were analyzed by immunoblotting with the indicated antibodies. Induction of apoptosis was evaluated by PARP cleavage. Actin was used as a loading control. Three representative cases are shown (G329, G333, G372). (B) Correlation between change in Mcl-1 expression and relative protection from ABT-199–induced apoptosis. The latter was defined as the increase in the percentage of viable cells treated with ABT-199 + imm-aIgM relative to ABT-199 alone (%Viable_{ABT-199+imm-aIgM} − %Viable_ABT-199)/%Viable_{ABT-199+imm-aIgM} × 100. Change in Mcl-1 expression was defined as fold change in imm-aIgM–stimulated/ABT-199–treated relative to unstimulated/ABT-199–treated CLL B cells. The relationship between Mcl-1 expression and the capacity of imm-aIgM to protect from ABT-199–induced apoptosis was evaluated through Pearson rank correlation. (C) Differences in expression of Bcl-2 family members between unstimulated/ABT-199–treated and imm-aIgM–stimulated/ABT-199–treated cells. Values represent relative protein levels for Mcl-1 (n = 25), Bcl-2 (n = 9), Bcl-_xL (n = 9), Bim_EL (n = 11) and Bim_L (n = 11), and relative mRNA levels for Bfl-1 (n = 7), Bmf (n = 8), and Hrk (n = 8). Antiapoptotic proteins are shown in the upper panels, and proapoptotic in the lower panels.