ShRNA-mediated knockdown of HOXA9 expression in human t(9;11) MOLM-14 and t(4;11) SEMK2 cells and murine leukemia stem cells (L-GMP). (A) Quantitative real-time PCR analysis of HOXA9 expression 72 hours after transduction of MOLM-14 and SEMK2 cells with 1 of 4 different HOXA9 shRNA constructs (1F2-HOXA9shRNA, 1F3-HOXA9shRNA, 2A4-HOXA9shRNA, and 2A5-HOXA9shRNA) without puromycin selection. A highly efficient HOXA9 suppression is demonstrated compared with a nontargeting control shRNA directed toward GFP. Western blot analysis 72 hours after transduction confirmed HOXA9 protein knockdown. (B) HoxA9 depletion in highly purified murine leukemia stem cells (L-GMP) resulted in a progressive induction of apoptosis in liquid culture compared with cells transduced with a control shRNA. (C) Analysis of L-GMP colony formation and replating capacity after HoxA9 suppression. Colonies were counted and replated at day 8. A significant decrease in colony number and replating capacity is demonstrated for L-GMPs transduced with HoxA9-directed shRNA without puromycin selection. (D) Analysis of L-GMP colony formation after HOXA9 suppression in a mixed-populations assay. L-GMPs were transduced with 1F2-HOXA9shRNA, 1F3-HOXA9shRNA, or GFP-control shRNA constructs, and populations of HOXA9shRNA or GFP-control shRNA-transduced cells were mixed in a ratio of 9:1 (HOXA9shRNA-transduced cells/GFP-control shRNA-transduced cells) and plated in semisolid medium. The total number of colonies at day 8 is shown for the mixed populations (1F2-HOXA9shRNA/GFP-control-shRNA; 1F3-HOXA9shRNA/GFP-control-shRNA) and the controls containing only GFP-control shRNA-transduced cells. (E) Quantitative real-time PCR further demonstrated similar HOXA9 expression in colonies that arose from the mixed HOXA9shRNA/GFP-control shRNA populations and controls containing only GFP-control shRNA-transduced cells (day 8).