Gene silencing of Bim or FOXO3a by siRNA prevents apoptosis induced by RTK inhibitor. (A,B) FDC-P1/FLT3-ITD cells were transfected by nucleofection with siRNA specific for mouse Bim (0.7μg) or with a control siRNA (pmaxGFP; 1.5 μg). Four hours after transfection, AG1295 was added as indicated. Cells were harvested after 24 hours and analyzed for effects on Bim expression by Western blot analysis (A) or apoptosis after annexin V–FITC/PI staining and flow cytometry (B). Data shown are mean (+ SD) from 3 individual experiments. (C-F) FOXO3a siRNA oligonucleotides were introduced to FDC-P1/FLT3-ITD cells by nucleofection. Cell lysates were prepared after 24 hours for Western blot analysis (n = 2). In separate experiments, the expression of Bim was analyzed by Western blot analysis in cells transfected with pmaxGFP, 1.3 μg FOXO3a, or Bim siRNA (D). The numbers of viable cells were analyzed by annexin V–FITC/PI staining and flow cytometry after treatment with either AG1295 at 5 μM (E) or PKC412 at 5 nM (F) for 24 hours. The inhibitors were added to cells after 4, 24, or 48 hours after transfection. Data represent mean (± SD) from 2 experiments.