Growth stimulatory and antiapoptotic signals in FLT3-ITD–transduced FDC-P1 cells is mediated by the PI-3 kinase/AKT pathway. FDC-P1 cells were retrovirally transduced with human FLT3-ITD. (A) Expression was confirmed by immunoprecipitation and immunoblotting for human FLT3. FDC-P1 cells expressing wild-type FLT3 or FLT3-ITD were cytokine-deprived and analyzed for survival during a 9-day incubation period by annexin V–FITC/PI staining and flow cytometry. One representative experiment of 3 performed yielding similar results is shown. (B) Whole-cell lysates were prepared from FDC-P1/FLT3 and FDC-P1/FLT3-ITD cells after 16 hours of cytokine deprivation followed by stimulation with 50 ng/mL FL, 10 ng/mL IL-3, or no cytokine addition. Western blot analysis was performed for p-AKT and p-FOXO3a. All blots were reprobed with a GAPDH antibody to demonstrate equal loading. (C) FDC-P1/FLT3-ITD cells treated with either 20 μM LY294002 or 50 μM PD98059 for 24 hours were stained for apoptotic cells with annexin V–FITC/PI and analyzed by flow cytometry. (D) Cell-cycle analysis was performed on FDC-P1/FLT3-ITD cells after 16 hours of treatment with either 20 μM LY294002 or 50 μM PD98059. Numbers of viable cells in S-phase were determined by flow cytometry. Bim expression was determined by Western blot analysis. Data are mean (± standard deviation [SD]) from 3 individual experiments performed in duplicates. **P < .01 (SD over control without inhibitor); ***P < .001 (SD).