Induced deletion of Ezh2 in MLL-AF9–transformed GMPs. (A) Schematic diagram of the experimental process. GMPs purified from Cre-ERT;Ezh2^+/+ or Cre-ERT;Ezh2^fl/fl mice were transduced with MLL-AF9 and cultured in methylcellulose medium. For deletion of Ezh2 in vitro, MLL-AF9–transformed GMPs were transferred to liquid medium containing 200nM 4-OHT. MLL-AF9–transformed GMPs were also transplanted into lethally irradiated recipient mice together with wild-type BM cells for radioprotection. For deletion of Ezh2 in vivo, 100 μL of tamoxifen (10 mg/mL) was IP injected once a day for 5 consecutive days at 3 weeks after transplantation. For serial transplantation assays, CD45.2⁺ leukemic cells were sorted from primary recipient mice with overt leukemia and transplanted into sublethally irradiated secondary recipient mice. (B) Efficient deletion of Ezh2 in MLL-AF9–transformed GMPs detected by genomic PCR. Representative genomic PCR showing the deletion of Ezh2 in Cre-ERT;Ezh2^fl/flMLL-AF9–transformed GMPs after culture in the presence of 200nM 4-OHT for 24 hours. “WT,” “floxed,” and “Δ” alleles indicate the wild-type allele, floxed Ezh2 allele, and floxed Ezh2 allele after removal of exons 18 and 19 by Cre recombinase, respectively. (C) Expression of Ezh2 in MLL-AF9–transformed GMPs in culture after Ezh2 deletion. mRNA levels of Ezh2 were evaluated by qRT-PCR and normalized to Hprt1 expression. Data are shown as the means ± SD for triplicate analyses. **P < .01. (D) Expression of MLL-AF9 in MLL-AF9–transformed GMPs in culture after Ezh2 deletion. mRNA levels of MLL-AF9 were evaluated by qRT-PCR and normalized to β-actin (Actb) expression. Data are shown as the means ± SD for triplicate analyses.