MLL-AF4 depletion inhibits colony formation and proliferation of t(4;11)-positive leukemic cells. (A) Specificity of MLL-AF4 and AML1/MTG8 siRNAs. SEM cells express MLL-AF4, whereas Kasumi-1 cells express AML1/MTG8. Colony numbers of siMA6-treated SEM cells are significantly lower than SEM controls (P < .001). (B) Inhibition of SEM and RS4;11 clonogenicity is dependent on perfect homology to the MLL-AF4 fusion site. Colony numbers of siMA6-treated SEM cells and siMARS-treated RS4;11 cells are significantly lower than the corresponding controls (P < .001). (C) MLL-AF4 siRNAs do not affect colony formation of primary human CD34⁺ hematopoietic cells. In all figure parts, colony formation is shown after electroporation with 750 nM siRNA. siMA6 indicates MLL-AF4 siRNA targeting the e9-e4 variant expressed in SEM; siMARS, MLL-AF4 siRNA targeting the e10-e4 variant expressed in RS4;11; siAGF1, AML1/MTG8 siRNA; siMM, siAGF6, mismatch control siRNAs. (D) Growth curves of siRNA-treated t(4;11) cell lines. Cells were electroporated every second day with 750 nM siRNA. Cell numbers were determined by MTT assays. (E) Effects of MLL-AF4 siRNAs on the cell-cycle distribution of SEM and RS4;11 cells. The graphs show the percentage of cells in the indicated cycle phase. Cell cycle distribution was determined by flow cytometry at the indicated days using cells from the time course experiments shown in panel D.