FOXO3a localization is not controlled by the ERK/MAPK signaling pathway. (A) After purification, AML blast cells were cultured during 4 hours in cytokine- and serum-free medium. During the last hour of starvation, cells were treated with or without UO126. MV4-11/FOXO3a-GFP cells were cultured in 10% FCS MEM and treated with UO126 during 1 hour. Protein extracts from 10⁶ cells were analyzed by Western blot. Phospho-ERK1/2 T²⁰²/Y²⁰⁴ was quantified and normalized to Actin signal intensity in 4 different AML samples. Results are expressed as a ratio to the control incubation without UO126. The statistical significance was calculated by Student t test, and vertical bars indicate SDs. (B) FOXO3a localization in both AML cells from 4 different AML samples and MV4-11/FOXO3a-GFP cells treated or not with IC87114 was investigated as described in Figure 1C. (C) 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. (D) Primary AML blast cells from patient 45 treated or not with UO126 were subjected to cellular fractionation as described in Figure 1E.