Effect of c-Myb inhibition on MLL-ENL mediated transformation. (A) A retroviral construct in a “self-inactivating” (SIN) viral backbone (pSIRENretroQ) expressing a small hairpin RNA (shRNA) specifically directing against c-Myb was tested for efficacy by cotransfection with a flag-c-Myb expression clone. A similar virus with a luciferase-directed shRNA sequence was used as control. shRNAs are processed in the cell to siRNA. Knockdown was verified by Western blotting with anti-flag antibodies (top panel) and by transduction of primary hematopoietic cells with Myb shRNA viruses followed by qRT-PCR of c-Myb RNA (bar graph gives mean and standard deviation of 3 samples). Primary hematopoietic precursors were simultaneously transduced with a pMSCV-MLL-ENL virus and either the luciferase or the c-Myb shRNA construct. The cells were tested for enhanced replicative capacity as a marker for transformation in a methocel serial-replating assay under appropriate antibiotic selection. First-round and third-round colonies are shown in a representative example of 3 independent experiments. A numeric evaluation of relative colony numbers (n = 3) is shown in the bottom panel. (B) In a parallel experiment, c-Myb activity was reduced by coexpression of a dominant-negative c-Myb derivative (DNmyb). For this purpose the DNA binding domain of c-Myb (amino acids 1-200) was fused to a KRAB repressor domain. Correct expression of the flag-tagged construct was verified by an anti-flag immunoblot. Representative colony formation of cells expressing MLL-ENL and DNmyb or as control MLL-ENL and flag-c-Myb is shown as in panel A. Colonies were phtographed with a Canon Coolpix 990 camera (Canon, Tokyo, Japan) in nanomode after staining with iodonitro tetrazolim violet. Image processing was done with standard Windows (Microsoft, Redmond, WA) processing software.