MLL-AF9 cells require DOT1L to maintain transformation in vitro. (A) Diagram of the procedure analyzing role of DOT1L in maintaining transformation in vitro. (B) Genotyping to verify efficient Cre-mediated recombination of DOT1L^2lox conditional allele. (C) RT-qPCR analysis of DOT1L expression level in 2lox/1lox MLL-AF9 cells, normalized to Gapdh, after 7 days of TAM treatment (250nM final concentration) demonstrates efficient recombination. (D) DOT1L is required for maintaining MLL-AF9 transformation but not for MLL-AFX. Methylcellulose colony formation assay after TAM treatment of transformed cells. Equal number of cells were plated and average numbers of CFUs from 3 independent experiments are shown. Error bars represent SD.