STAT5 is not required for B-lymphoid transformation by BCR-ABL1 in vitro or for maintanance of established B-lymphoid leukemia in viro. (A-B) STAT5 is not required for transformation of primary BM B-lymphoid cells by BCR-ABL1 in vitro. (A) Primary BM from non–5-FU-treated Stat5a/b^fl/+ and Stat5a/b^fl/− donor mice was transduced with p210MIGFP or p210MIGFPCre retrovirus and plated directly in agarose. Transformed pre-B lymphoid colonies were counted on day 10. There was no significant difference in the number of colonies arising from Stat5a/b^fl/− donor BM transduced with either p210MIGFP or p210MIGFPCre retrovirus (P = .88) or between any of the values assessed pairwise (t tests). (B) Serial dilutions of transduced BM were plated in triplicate on syngeneic stromal layers derived from nontransduced wild-type BM and cultured for 3 weeks, as described in the “Methods.”²⁹  The plating density is indicated by the line color, the number of cultures that reached confluence (defined as 10⁶ nonadherent cells) is indicated on the ordinate, and the time to confluence on the abscissa. (C) Growth of BCR-ABL1–transformed B-lymphoblasts derived from Mx-Cre;Stat5a/b^fl/+ (top panel) or Mx-Cre;Stat5a/b^fl/− (bottom panel) donors either untreated (blue squares) or treated with IFN-β (red circles). Each curve represents data from an independent population of transformed cells. (D) Southern blot analysis of Stat5a/b recombination status from the experiment in panel C. Lanes 1 and 2 contain tail DNA from Stat5 wild-type and Stat5^fl/− mice, respectively. Note the complete deletion of the floxed Stat5a/b allele in IFN-treated lymphoblasts from Mx-Cre;Stat5a/b^fl/+ donors (lanes 7-10) and from Mx-Cre;Stat5a/b^fl/− donors (lanes 15-18). (E) Kaplan-Meier survival curve for unirradiated Balb/c Rag2^−/− recipients injected intravenously (1 × 10⁷ cells each, n = 16) with BCR-ABL1–expressing lymphoblasts derived from the in vitro transformation experiments in panels B and C. After engraftment of leukemia, half of each cohort (dashed lines) were treated with pIpC (arrowheads) to induce Cre recombinase. (F) Southern blot analysis of Stat5a/b recombination status in tumor tissue from 3 representative recipients of lymphoblasts from Mx-Cre;Stat5a/b^fl/− donors from panel E that were treated with pIpC (lanes 4-6) or untreated (lanes 7-9). Lanes 1, 2, and 3 contain tail DNA from Stat5wt, Stat5^fl/+, and Stat5^fl/− mice, respectively. Note the complete deletion of the floxed Stat5a/b allele in lymphoblasts from pIpC-treated recipients.