Dot1l is required for transformation by MLL-AF9 but not by Hoxa9/Meis1 and E2A-HLF. (A) Schematic of CFU assay. Dot1l^wt/wt;CreER⁺ (Dot1l^+/+) or Dot1l^F/F;CreER⁺ (Dot1l^F/F) mice were injected with 5-fluorouracil, and 4 days later bone marrow cells were harvested. The cells were retrovirally transduced with indicated oncogenes (Hoxa9/Meis1, E2A-HLF, or MLL-AF9) and plated on methocult media for 3 rounds either with 4-OHT for Dot1l excision or solvent ethanol (EtOH) as control. (B) Genotyping of transduced bone marrow cells after the second round. PCR reaction showed high excision efficiency with 4-OHT treatment in all cells. (C) Colony formation on methocult plates. Dot1l^F/F MLL-AF9 cells treated with 4-OHT failed to form colonies. (D) Bar graph of colony count at final round. Data are mean ± SD.