Role of Setdb1 in leukemic stem cells. (A) A schematic diagram of the experimental process. GMPs from CreERT and CreERT;Setdb1^fl/fl mice were transduced with MLL-AF9 and cultured in methylcellulose medium. To delete Setdb1 in vitro, MLL-AF9-transformed GMPs were transferred to liquid medium containing 200 nM 4-hydroxy tamoxifen (4-OHT). MLL-AF9-transformed GMPs were also transplanted into lethally irradiated recipient mice, together with WT BM cells for radioprotection. To delete Setdb1 in vivo, tamoxifen was intraperitoneally injected once a day for 5 consecutive days at 21 days after transplantation. (B) Growth of MLL-AF9-transformed GMPs after the deletion of Setdb1. MLL-AF9-transformed GMPs (1 × 10⁴ cells each) were cultured in IMDM with 20% fetal calf serum, SCF, FP6, GM-CSF, and IL-3 (10 ng/mL each). Data are shown as the mean ± SD of triplicate cultures. (C) Replating efficiency of MLL-AF9-transformed GMPs after the deletion of Setdb1. MLL-AF9-transformed GMPs (1500 cells) were serially replated in methylcellulose medium containing 10 ng/mL SCF, 10 ng/mL FP6, 10 ng/mL GM-CSF, 10 ng/mL IL-3, and 100 nM 4-OHT. Data are shown as the mean ± SD of triplicate cultures. (D) Overall survival of mice injected with 4 × 10⁵ WT or Setdb1^Δ/ΔMLL-AF9-transformed cells compared by a Kaplan-Meier analysis (WT, n = 10; Setdb1^Δ/Δ n = 9). (E) The efficiency of the deletion of Setdb1 in MLL-AF9-transformed GMPs was monitored. Genomic PCR data of leukemic cells immediately after the injection of tamoxifen and from moribund mice (#1 and #2, respectively, indicated in panel D). *P < .05; **P < .01; ***P < .001.