Constitutively active FOXO3a (FOXO3a-AAA) inhibits the proliferation of NPM-ALK-transformed Ba/F3 cells. (A) Expression of NPM-ALK in TonBaF.1-FOXO3a and TonBaF.1-FOXO3a-AAA cells stably transformed with NPM-ALK. Whole-cell lysates were fractionated by SDS-PAGE and immunoblotted with ALK-specific antibody. The minus signs represent parental TonBaF.1 cells. (B) Expression of FOXO3a or FOXO3a-AAA in TonBaF.1-FOXO3a and TonBaF.1-FOXO3a-AAA cells stably transformed with NPM-ALK after 72-hour incubation with 2 μg/mL doxycycline. Whole-cell lysates were fractionated by SDS-PAGE and immunoblotted with antiphospho-FOXO3a (Thr32)-specific antibody. The membrane was stripped and reprobed with anti-FOXO3a-specific antibody. (C) Effect of FOXO3a or FOXO3a-AAA expression on the proliferation of NPM-ALK-transformed Ba/F3 cells. Thymidine incorporation assay was performed after 72 hours of culture. Values represent the mean ± SD of triplicate cultures and are expressed as percentages of uninduced control cells. (D) Western blot analysis of p27^kip1 expression in TonBaF.1-FOXO3a or TonBaF.1-FOXO3a-AAA cells stably transformed with NPM-ALK. Whole-cell lysates were harvested and fractionated by SDS-PAGE and immunoblotted with p27^kip1-specific antibody. The membrane was stripped and reprobed with Rack1-specific antibody to confirm equal loading.