Mcl-1 mediates ABT-199 resistance. (A-C) CLL cells were transfected with control (siCont) or Mcl-1 (siMcl-1) siRNA, split and cultured with or without imm-aIgM for 3 hours prior to the addition of ABT-199 (2 nM). The levels of Mcl-1 protein were evaluated by immunoblotting of cellular extracts collected 3 and 18 hours after the addition of imm-aIgM. The percentage of viable cells was determined by annexin V/PI staining 20 hours after the addition of imm-aIgM. One representative experiment is shown in panels A and B, respectively. Summary of the data from 5 independent experiments evaluating leukemic cell viability is shown in panel C. Significance of differences in leukemic cell viability was evaluated with the paired Student t test. (D-F) CLL cells were transfected with Mcl-1 or control mRNA. ABT-199 was added after 3 hours, when Mcl-1 protein levels reached maximum, and was removed 3 hours later when Mcl-1 protein levels started to substantially decline (Mcl-1 protein half-life is <1 hour). The ABT-199 concentration in these experiments was increased from 2 nM to 4 nM because of the shorter exposure to the drug. Levels of Mcl-1 protein in CLL cells collected 3 hours postnucleofection are shown in panel D. Analysis of leukemic cell viability in 1 representative sample 20 hours after nucleofection is shown in panel E, whereas summary of data from 5 different experiments evaluating leukemic cell viability is shown in panel F. Significance of differences was evaluated with the paired Student t test.