Analysis of phenotypic consequences after shRNA-mediated HOXA9 suppression in t(9;11) MOLM-14 AML and t(4;11) SEMK2 ALL cells. (A) Analysis of cell viability as measured by annexin V/PI positivity at day 7 after transduction of t(9,11) MOLM-14 cells and t(4;11) SEMK2 cells with 1 of 4 HOXA9 targeted shRNAs or GFP control shRNA. The percentage of viable (annexin V/PI negative) cells is shown in the HOXA9shRNA and GFP control shRNA-transduced group. (B) Time course analysis of cell viability as measured by annexin V/PI positivity after HOXA9 suppression in an independent experiment. Cells were transduced with either 1 of 4 HOXA9 targeted shRNAs or GFP-control shRNA. The data are graphed as percentage of viable cells that were transduced with HOXA9shRNA/percentage of viable cells that were transduced with GFP-control shRNA at a given time point. These results were confirmed 3 times. (C) Quantitative real-time PCR and Western blot analysis of ectopic HOXA9 overexpression in t(9;11) MOLM-14 cells compared with untransduced cells and cells transduced with the empty vector construct. (D) MOLM-14 cells ectopically overexpressing HOXA9 were transduced with the 2A4-HOXA9shRNA construct specifically targeting the 3′-untranslated region of endogenous HOXA9 or the GFP-control shRNA construct. Ectopic overexpression of HOXA9 almost completely rescued the phenotype (annexin V/PI staining).