Requirement for MCL-1 during in vivo BCR-ABL leukemia maintenance. (A) Mcl-1^f/f Rosa-ERCre^T2 (Arf^wt) or Mcl-1^wt Rosa-ERCre^T2 BM was transduced with p185-IRES-GFP and transplanted into lethally irradiated (1100 rad) C57BL/6 recipients that were monitored for leukemia initiation. After leukemia initiation, BM from the leukemic mice (2 independent leukemia donors per genotype) was harvested and mixed 1:1 with control CD45.1 congenic BM and transplanted into secondary, lethally irradiated (1100 rad) C57BL/6 recipients (∼10 secondary recipients each for 2 separate leukemic donors). After 5 days, the secondary recipients were treated with 5 daily doses of TAM (1 µg/d) to activate Cre (n = 13 mice for Mcl-1^f/f Rosa-ERCre^T2 and n = 10 for Mcl-1^wt Rosa-ERCre^T2 donors) or control vehicle (n = 9 mice for Mcl-1^f/f Rosa-ERCre^T2 and n = 9 for Mcl-1^wt Rosa-ERCre^T2 donors) by gavage. Mice were monitored daily and euthanized when moribund. Asterisk (*) denotes P < .001 by log-rank test. (B) White blood cell (WBC) counts were taken from moribund, secondary recipients described in panel A at the time of being euthanized. Bars indicate the averages (n = 6 mice for TAM-treated Mcl-1^f/f Rosa-ERCre^T2 donors, n = 9 for TAM-treated Mcl-1^wt Rosa-ERCre^T2 donors, n = 9 mice for vehicle-treated Mcl-1^f/f Rosa-ERCre^T2 donors, and n = 9 for vehicle-treated Mcl-1^wt Rosa-ERCre^T2 donors), and error bars indicate SEM. (C) Representative immunoblot shown of GFP⁺ BM isolated from TAM-treated, moribund mice that received p185⁺Mcl-1^f/f Rosa-ERCre^T2 leukemic BM (each lane represents a recipient mouse). Lysates were blotted for MCL-1, BCR-ABL (as detected by anti-ABL antibody), and ERCre^T2 protein as a loading control (note: ERCre^T2 fusion protein is present in all cells but is only activated by TAM). (D) Representative Mcl-1, Mcl-1–deleted, and Cre genotyping of GFP⁺ BM isolated from TAM-treated, moribund mice that received p185⁺Mcl-1^f/f Rosa-ERCre^T2 leukemic BM (each lane represents a recipient mouse). (E) Histopathological examination of moribund vehicle-treated or TAM-treated mice that received p185⁺Mcl-1^f/f Rosa-ERCre^T2 leukemic BM. Images of indicated tissues (×50 magnification) are representative of 9 vehicle-treated mice and 4 moribund, TAM-treated mice analyzed. Hematoxylin and eosin (H&E) and PAX5 immunohistochemistry are morphologically and immunophenotypically indicative of B-lineage leukemia.