Nuclear FOXO1 and PI3K activity coexist in BL cells. (A) Immunofluorescence analysis of human BL cell lines using FOXO1 antibody (red). Cell nuclei were counterstained with DAPI (blue; scale bar, 5 μm). (B) Western blot analysis of FOXO1 expression in subcellular fractions of human BL cells. The purity of the cytoplasmic and nuclear fraction was determined by actin (ACTB) and histone H3 (H3) antibodies, respectively. Data are representative of at least 2 experiments. (C) Immunofluorescence analysis of FOXO1 in mouse BL cell lines as shown in panel A. (D) Western blot analysis of phospho-AKT (pAKT [S473]), AKT, phospho-FOXO1 (pFOXO1 [T24]), and FOXO1 expression in human BL cell lines. Cells were treated either with the PI3K inhibitor wortmannin (+) or dimethyl sulfoxide (DMSO) (−) 1 hour prior to protein extraction. ACTB served as loading control. Data are representative of 2 experiments. (E) Detection of pAKT(S473) in mouse BL-like cell lines by intracellular FACS analysis. After fixation and permeabilization, lymphoma cells were incubated with pAKT(S473) antibody (= antibody) or the antibody plus pAKT(S473) blocking peptide (= blocking peptide). Data are representative of 2 experiments. C, cytoplasmic fraction; KO, FOXO1 knockout cell; N, nuclear fraction; W, whole-cell lysate.