Requirement for MCL-1 during in vitro and in vivo BCR-ABL leukemia initiation. (A-B) BM from wild-type Mcl-1 (Mcl-1^wt), Mcl-1^f/wt, or Mcl-l^f/f mice on Arf^wt (A) or Arf^−/− (B) genetic backgrounds was transduced with p185-IRES-Cre retrovirus to transform and delete the floxed allele. Viable cell concentration was assessed every 24 hours. Each time point represents an average of 3 independent experiments (n = 3), and the error bars denote SEM. Data represent 10 separate cultures from 2 independent mice. (C-D) Representative Mcl-1, Mcl-1–deleted, and Cre genomic genotyping preformed post outgrowth from Arf^wt (C) and Arf^−/− (D) genetic backgrounds. (E) Immunoblot analysis from cells that grew out in culture from the Arf^−/− experiment detailed in (B) represent 2 independent experiments. Lysates were blotted for MCL-1, BCR-ABL (as detected by anti-ABL antibody), Cre, and Ponceau stain (loading control). (F) Arf^−/− BM from Mcl-1^wt (n = 10 mice), Mcl-1^f/wt (n = 10 mice), or Mcl-1^f/f (n = 10 mice) were transduced with p185-IRES-Cre retrovirus and immediately transplanted into lethally irradiated (1100 rad) C57BL/6 recipients. Data represent 10 individual leukemia recipients. BM from 2 independent donor mice was used. Recipient mice where monitored daily and euthanized when moribund. Asterisk (*) denotes P < .001 by log-rank test when compared with Mcl-1^wt. (G) Whole blood cell (WBC) counts were taken from each mouse at the time of being euthanized. Each point represents 1 mouse, the horizontal line indicates the averages, and the error bars represent SEM. (H) Representative Mcl-1, deleted Mcl-1, and Cre genotyping of BM cells isolated from 2 representative moribund mice. (I) Representative immunoblots of BM from 3 individual moribund mice were blotted for MCL-1, Cre, and actin (loading control).