T24 mutation or NES deficiency of FOXO1 act redundantly on mouse BL growth. (A) Schematic depiction of CRISPR/Cas9 targeting at the endogenous mouse Foxo1 locus to repair the T24 mutation. The Foxo1 gRNA_e was selected to specifically target the mt allele because of the lack of the PAM sequence at the wt allele. (B) Immunofluorescence analysis of mouse BL cells using FOXO1 antibody (red). Parental cells (mouse BL#19) carrying a heterozygous Foxo1 T24 mutation (wt⁺/mt⁺) and an isogenic cell line clone in which the mt allele was repaired to express exclusively wt FOXO1 (wt⁺/wt^rep) are shown. Cell nuclei were counterstained with DAPI (blue; scale bar, 10 μm). (C) Growth curves of parental cells (wt⁺/mt⁺) and isogenic cell line clones (wt⁺/wt^rep) in which the mt allele was repaired by CRISPR/Cas9 genome editing. The graph summarizes data of 3 experiments. Bars indicate the standard deviation. ***P < .001 (Wilcoxon-Mann-Whitney test). (D) Murine stem cell virus–based vector constructs for overexpression of wt and mt FOXO1. Transgene expression is coupled to BFP via an internal ribosome entry site sequence. (E) Immunofluorescence analysis of infected mouse BL cells using FOXO1 antibody (red). Foxo1 KO cells were either transduced with an empty vector (BFP) or the constructs described in panel D. Cell nuclei were counterstained with DAPI (blue; scale bar, 10 μm). (F) Percentage of BFP expressing cells over time. After infection of Foxo1 KO cells with the constructs described in panel D, the proportion of transgene-expressing cells was monitored by FACS at indicated time points. The graph summarizes the results of 2 infections. Bars indicate the standard deviation. ***P < .001; ****P < .0001 (Wilcoxon-Mann-Whitney test). (G) Western blot analysis of AKT-dependent FOXO1 phosphorylation in mouse BL cells infected with the constructs depicted in panel D. Cells expressing mt FOXO1 AAA (FOXO1^3A), which is impaired in T24, S256, and S319 phosphorylation, were included as controls. ACTB served as loading control. Data are representative of 2 experiments.