FOXO3a is the prevalent active FOXO in AML blast cells. (A) BM blast cells from 7 patients were highly purified by flow cytometric cell sorting according to CD45low expression and side scatter. FOXO1, FOXO3a, and FOXO4 mRNA levels were quantified by RT-qPCR. Their levels were expressed relative to HPRT mRNA level. (B) Expression of FOXO1, FOXO3a, and FOXO4 in total cell lysates from 9 AML samples was compared with human leukemic cell lines (MOLM-14 and MV4-11). Protein extracts from 106 cells were analyzed by Western blot. (C) FOXO3a localization in AML cells from 17 different AML samples was investigated using IF staining (green: FOXO3a; blue: nuclear DAPI). Images obtained from representative patients 17 and 25 are presented. FOXO3a localization in MV4-11/FOXO3a-GFP cells was investigated by direct analysis of subcellular GFP distribution. Three-dimensional image of the fluorescence intensity of indicated cell is reported. (D) For the quantification of the percentage of cells with nuclear FOXO3a, 100 cells were counted. The statistical significance was calculated by Student t test, and vertical bars indicate SDs. (E) Primary AML blast cells from patients 290 and 31 were subjected to cellular fractionation. Protein extracts from 106 cells were analyzed by Western blot. N and C indicate nuclear and cytoplasmic fraction, respectively.
