DOT1L is required for MLL-AF9-induced transformation in vitro. (A) Diagram of the procedure used for in vitro analysis. (B) Bone marrow cells were stained with rat IgG2a-APC isotype control to identify c-Kit-negative population or rat antimouse c-Kit-APC. c-Kit⁺ cells were sorted using a BD FACSAria II instrument. (C) RT-qPCR analysis of DOT1L expression level, normalized to Gapdh, after 7 days of TAM treatment (250nM final concentration) demonstrates efficient recombination. Mouse genotype is indicated. (D) Serial methylcellulose colony replating assay shows that MLL-AF9 fails to transform HPCs in the absence of DOT1L. An equal number of cells transduced with empty vector MSCN or MLL-AF9 were plated at each round and colony forming units (CFUs) counted after 7 to10 days. Experiment was performed 3 times and presented as average number of CFUs with standard deviation (SD).