American Society of Hematology

Figure 2.

$Figure 2. Mutations impairing T24 phosphorylation lock FOXO1 in the cell nucleus. (A) Pie charts indicating the incidence of nonsynonymous FOXO1 mutations in human BL cell lines (left) and mouse BL-like tumors (right). The bar diagrams show the percentage of FOXO1 mutations involving the AKT-dependent phosphorylation site at T24 (T24) or other positions (others) in human and mouse BL. (B) Design of FOXO1 gRNAs used for CRISPR/Cas9 experiments in human and mouse BL cells. CRISPR/Cas9 target sequences are given in blue (protospacer) and red (PAM). In the mouse FOXO1 locus the nucleotide affected by the T24 mutation is marked in yellow. (C) Experimental scheme for the generation of isogenic cell line clones analyzed in panels D-F. After electroporation and transient coexpression of gRNA, Cas9 and mCherry individual reporter-positive cells were FACS-based sorted and expanded for analysis. (D) Immunofluorescence analysis of FOXO1 (red) in CRISPR/Cas9-modified human and mouse BL cells. Parental cell lines (BL2 and mouse BL#19) carrying heterozygous FOXO1 T24 mutations (wt+/mt+) and isogenic cell line clones in which the mt alleles were ablated (wt+/mt−) are shown. Cell nuclei were counterstained with DAPI (blue; scale bar, 5 μm). (E) Western blot analysis of FOXO1 expression in subcellular fractions of mouse BL#19 cells (wt+/mt+) and isogenic cell line clones in which the wt (wt−/mt+) or the mt Foxo1 allele (wt+/mt−) was specifically ablated. The purity of the cytoplasmic and nuclear fraction was determined by actin (ACTB) and histone H3 (H3) antibodies, respectively. In addition, pFOXO1 (T24), pAKT (S473), and AKT expression were detected by the appropriate antibodies. Data are representative of 2 experiments. (F) Western blot analysis of 14-3-3 protein expression after immunoprecipitation with FOXO1 antibody in isogenic mouse BL cell lines (as described in panel D). Per genotype, 3 individual cell line clones were analyzed. The membrane was also incubated with anti-FOXO1 antibody to verify precipitation of the transcription factor. Data are representative of 2 experiments. C, cytoplasmic fraction; N, nuclear fraction.$

Mutations impairing T24 phosphorylation lock FOXO1 in the cell nucleus. (A) Pie charts indicating the incidence of nonsynonymous FOXO1 mutations in human BL cell lines (left) and mouse BL-like tumors (right). The bar diagrams show the percentage of FOXO1 mutations involving the AKT-dependent phosphorylation site at T24 (T24) or other positions (others) in human and mouse BL. (B) Design of FOXO1 gRNAs used for CRISPR/Cas9 experiments in human and mouse BL cells. CRISPR/Cas9 target sequences are given in blue (protospacer) and red (PAM). In the mouse FOXO1 locus the nucleotide affected by the T24 mutation is marked in yellow. (C) Experimental scheme for the generation of isogenic cell line clones analyzed in panels D-F. After electroporation and transient coexpression of gRNA, Cas9 and mCherry individual reporter-positive cells were FACS-based sorted and expanded for analysis. (D) Immunofluorescence analysis of FOXO1 (red) in CRISPR/Cas9-modified human and mouse BL cells. Parental cell lines (BL2 and mouse BL#19) carrying heterozygous FOXO1 T24 mutations (wt⁺/mt⁺) and isogenic cell line clones in which the mt alleles were ablated (wt⁺/mt⁻) are shown. Cell nuclei were counterstained with DAPI (blue; scale bar, 5 μm). (E) Western blot analysis of FOXO1 expression in subcellular fractions of mouse BL#19 cells (wt⁺/mt⁺) and isogenic cell line clones in which the wt (wt⁻/mt⁺) or the mt Foxo1 allele (wt⁺/mt⁻) was specifically ablated. The purity of the cytoplasmic and nuclear fraction was determined by actin (ACTB) and histone H3 (H3) antibodies, respectively. In addition, pFOXO1 (T24), pAKT (S473), and AKT expression were detected by the appropriate antibodies. Data are representative of 2 experiments. (F) Western blot analysis of 14-3-3 protein expression after immunoprecipitation with FOXO1 antibody in isogenic mouse BL cell lines (as described in panel D). Per genotype, 3 individual cell line clones were analyzed. The membrane was also incubated with anti-FOXO1 antibody to verify precipitation of the transcription factor. Data are representative of 2 experiments. C, cytoplasmic fraction; N, nuclear fraction.

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