Inhibition of FLT3-ITD signaling by AG1295 or PKC412 leads to dephosphorylation of FOXO3a and up-regulation of proapopotic Bim and Puma. FDC-P1/FLT3-ITD cells were treated for 24 hours with increasing concentrations of AG1295 (A) or PKC412 (B), then whole-cell lysates were prepared and analyzed for expression of Bim, Puma, and caspase-3. (C) FDC-P1/FLT3-ITD cells were treated with AG1295, and the amount of p-FOXO3a and expression of Bim, Puma, and caspase-3 was analyzed by Western blot analysis (n = 3). Anti-GAPDH antibody was used as a loading control. (D-F) FDC-P1/FLT3-ITD cells infected with pBabe:FOXO3(A3):ER were stimulated with 100 nM 4-OHT. After 8 and 24 hours, cells were analyzed for DNA content by PI staining and flow cytometry. Results are mean (± SD) from 2 experiments (D). Cells were also analyzed for apoptosis by flow cytometry after staining with annexin V–FITC/PI. Results are mean (± SD) from duplicates and repeated twice. **P < .01 (SD over control; E). Cell lysates were prepared at times indicated, and Western blot analysis was performed using Bim, Puma, or GAPDH antibodies (n = 2; F).