Dot1l excision leads to cell cycle defect in cells with MLL-AF9 oncogene and to the loss of critical downstream target gene expression. (A) Growth curve of Dot1l^wt/wt;CreER⁺ (Dot1l^+/+) or Dot1l^F/F;CreER⁺ (Dot1l^F/F) cells transduced with Hoxa9/Meis1, E2A-HLF, or MLL-AF9. Cells were grown in culture for more than 8 weeks. Cell growth was measured by CellTiter-Glow assay with either EtOH or 4-OHT treatment. MLL-AF9–transformed cells failed to grow in the absence of Dot1l, whereas Hoxa9/Meis1 and E2A-HLF transformed cells continued to grow. All values were normalized to day 1. Data are mean ± SD. (B) H3K79me2 Western blot showed loss of methylation with 4-OHT treatment in all Dot1l^F/F cell lines. Whole cell lysate samples were harvested 4 days after EtOH or 4-OHT treatment. Histone 3 blot was used as loading control. (C) Quantification of cell cycle. Cells were stained with propidium iodide 4 days after EtOH or 4-OHT treatment. Values are ratio of 4-OHT treatment over EtOH treatment for respective cell lines. Dot1l^F/F MLL-AF9 cell line showed increase in G₀-G₁ and decrease in S and G₂-M phases with 4-OHT treatment. Data are mean ± SD. (D) Staining profile of propidium iodide. (E) Quantitative PCR of Hoxa9 and Meis1 expression. Samples were collected 4 days after EtOH or 4-OHT treatment. MLL-AF9 cells showed loss of Hoxa9 and Meis1 expression with Dot1l excision. Values were normalized to 5S rRNA internal control and EtOH treatment for respective cell lines. Data are mean ± SD.