ASB2 reduces colony formation of MLL-AF9 transformed cells. (A) Colony numbers of MLL-AF9 cells transduced with empty vector or ASB2 from methylcellulose replating assay. Error bars indicate SD from 3 independent experiments. (B) P-iodonitro-tetrazolium violet staining of the colonies. (C) MLL-AF9 cells transduced with empty vector or ASB2 were grown in liquid culture. Significantly reduced cell proliferation was observed for ASB2-transduced cells. Error bars indicate SD. (D) RT-qPCR was performed to measure the expression of Hoxa9 in MLL-AF9 cells transduced with empty vector or ASB2. Expression is shown relative to empty vector-transduced control cells. Error bars indicate SD. (E) Schematic diagram showing the endogenous Hoxa9 locus and probes used in the ChIP assay. TSS: transcription start site. (F-H) ChIP assays were performed to determine the recruitment of Mll (F), the H3K4me3 level (G), and the recruitment of MLL-AF9 (H) on the Hoxa9 locus with probes shown in (E). Antibodies against MLL, H3K4me3 and Flag were used, respectively. Blue lines are data from MLL-AF9 cells transduced with empty vector; red lines are data from MLL-AF9 cells transduced with ASB2; and gray lines are IgG controls for each cell line. Error bars indicate SD.