Regulation of cytokine deprivation–induced apoptosis in mast cells is FOXO3a dependent. Survival of wt or FOXO3a^−/− mast cells in the absence of cytokines was measured by staining with PI plus annexin V-FITC. (A) MLMCs, where ◇ indicates wt and ■, FOXO3a^−/−. (B) CTLMCs, where ◇ indicates wt and ■, FOXO3a^−/−. N = 3-6 and results are presented as mean (± SD). (C) Expression of an inducible, dominant-active mutant of FOXO3 results in up-regulation of Puma and Bim. BMMCs were infected with a retrovirus encoding FOXO3(A3):ER and incubated with 4-hydroxy tamoxifen (4-OHT) (+) or left untreated (−) in the presence of SCF for 48 hours. The level of Puma or Bim was analyzed by Western blotting. As a control, uninfected cells were also treated with 4-OHT. Probing for Erk served as a loading control. (D) Expression of Puma and Bim was determined by Western blotting of lysates from FOXO3a^−/− MLMCs and CTLMCs following cytokine deprivation for 10 hours. Probing for Hsp70 (left panel) or beta-Actin (right panel) served as a loading control. The results shown (C,D) are representative of at least 3 independent experiments.