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foxo1 af647 (Cell Signaling Technology Inc)

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Sequential mutagenesis at the endogenous <t>Foxo1</t> locus. (A) Targeting strategy at the endogenous Foxo1 locus. A STOP cassette flanked by rox sites was introduced in front of the endogenous Kozak sequence, and the sequence of exon 1 was modified in order to contain a synonymous PAM mutation in position g.249G>A, which abrogated the genetic editing of the targeting vector used for the homologous recombination. The gRNA sequence used is shown in blue. Heterozygous mutant mice were verified by PCR and Sanger sequencing that allowed for the detection of the PAM mutation and thus verification of successful targeting. (B and C) Percentage of total GCBCs (B) and ZsGreen + GCBCs (C) from control Foxo1 fl/wt and experimental Foxo1 fl/stopRox mice at day 14 (TAM day 10–12) after immunization. (D) Pie charts showing the mutational load at day 14 (TAM day 10–12) after immunization of Foxo1 fl/wt control, Foxo1 fl/stopRox experimental, and Foxo1 fl/stopRox KO (without TAM administration) ZsGreen + GCBCs (for Foxo1 KO condition, total GCBCs were sorted) from 3 to 5 mice per condition. Number inside the pie chart indicates total number of sequences analyzed. (E) Experimental scheme for the analysis of CSR events in vitro , inducing Dre recombination (medium supplemented with 4-OHT) at day (d)0 (early time point) or day 4 (late time point) of coculture with 40LB feeder cells (+IL-4), and analyzing at day 4 or day 8, respectively. (F) Left panel: representative flow cytometry histogram showing Foxo1 intracellular expression in Foxo1 WT hom ( Foxo1 wt/wt —blue) and Foxo1 WT het ( Foxo1 fl/wt —brown) iGCBCs at day 4 of coculture. Foxo1 FMO is depicted in gray. Right panel: quantification of the MFI of Foxo1 subtracting the FMO. (G) Percentage of IgG1 + (left) or IgE + (right) iGCBCs at day 4 in Foxo1 WT cells. (H and I) Percentage of ZsGreen + iGCBCs (iGCs; left panel), percentage of IgG1 + and IgE + iGCBCs (middle panel), and Foxo1 MFI in iGCBCs (right panel) at (H) day 4—early time point—or at (I) day 8—late time point—of analysis. (J) Representative flow cytometry histogram showing Foxo1 intracellular expression in EtOH-treated (KO) vs. 4-OHT–treated (rescued) iGCBCs from experimental mice. Statistics: Mann–Whitney test, *P ≤ 0.05; **P ≤ 0.01; ns, not significant. Each dot represents one mouse. Data are from at least two independent experiments. Horizontal lines indicate the median. FMO, fluorescence minus one; MFI, median fluorescence intensity.

Foxo1 Af647, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Sequential mutagenesis at the endogenous Foxo1 locus. (A) Targeting strategy at the endogenous Foxo1 locus. A STOP cassette flanked by rox sites was introduced in front of the endogenous Kozak sequence, and the sequence of exon 1 was modified in order to contain a synonymous PAM mutation in position g.249G>A, which abrogated the genetic editing of the targeting vector used for the homologous recombination. The gRNA sequence used is shown in blue. Heterozygous mutant mice were verified by PCR and Sanger sequencing that allowed for the detection of the PAM mutation and thus verification of successful targeting. (B and C) Percentage of total GCBCs (B) and ZsGreen + GCBCs (C) from control Foxo1 fl/wt and experimental Foxo1 fl/stopRox mice at day 14 (TAM day 10–12) after immunization. (D) Pie charts showing the mutational load at day 14 (TAM day 10–12) after immunization of Foxo1 fl/wt control, Foxo1 fl/stopRox experimental, and Foxo1 fl/stopRox KO (without TAM administration) ZsGreen + GCBCs (for Foxo1 KO condition, total GCBCs were sorted) from 3 to 5 mice per condition. Number inside the pie chart indicates total number of sequences analyzed. (E) Experimental scheme for the analysis of CSR events in vitro , inducing Dre recombination (medium supplemented with 4-OHT) at day (d)0 (early time point) or day 4 (late time point) of coculture with 40LB feeder cells (+IL-4), and analyzing at day 4 or day 8, respectively. (F) Left panel: representative flow cytometry histogram showing Foxo1 intracellular expression in Foxo1 WT hom ( Foxo1 wt/wt —blue) and Foxo1 WT het ( Foxo1 fl/wt —brown) iGCBCs at day 4 of coculture. Foxo1 FMO is depicted in gray. Right panel: quantification of the MFI of Foxo1 subtracting the FMO. (G) Percentage of IgG1 + (left) or IgE + (right) iGCBCs at day 4 in Foxo1 WT cells. (H and I) Percentage of ZsGreen + iGCBCs (iGCs; left panel), percentage of IgG1 + and IgE + iGCBCs (middle panel), and Foxo1 MFI in iGCBCs (right panel) at (H) day 4—early time point—or at (I) day 8—late time point—of analysis. (J) Representative flow cytometry histogram showing Foxo1 intracellular expression in EtOH-treated (KO) vs. 4-OHT–treated (rescued) iGCBCs from experimental mice. Statistics: Mann–Whitney test, *P ≤ 0.05; **P ≤ 0.01; ns, not significant. Each dot represents one mouse. Data are from at least two independent experiments. Horizontal lines indicate the median. FMO, fluorescence minus one; MFI, median fluorescence intensity.

Journal: The Journal of Experimental Medicine

Article Title: FOXO1 re-expression with a dual-recombinase allele rescues class switching in germinal center B cells

doi: 10.1084/jem.20241136

Figure Lengend Snippet: Sequential mutagenesis at the endogenous Foxo1 locus. (A) Targeting strategy at the endogenous Foxo1 locus. A STOP cassette flanked by rox sites was introduced in front of the endogenous Kozak sequence, and the sequence of exon 1 was modified in order to contain a synonymous PAM mutation in position g.249G>A, which abrogated the genetic editing of the targeting vector used for the homologous recombination. The gRNA sequence used is shown in blue. Heterozygous mutant mice were verified by PCR and Sanger sequencing that allowed for the detection of the PAM mutation and thus verification of successful targeting. (B and C) Percentage of total GCBCs (B) and ZsGreen + GCBCs (C) from control Foxo1 fl/wt and experimental Foxo1 fl/stopRox mice at day 14 (TAM day 10–12) after immunization. (D) Pie charts showing the mutational load at day 14 (TAM day 10–12) after immunization of Foxo1 fl/wt control, Foxo1 fl/stopRox experimental, and Foxo1 fl/stopRox KO (without TAM administration) ZsGreen + GCBCs (for Foxo1 KO condition, total GCBCs were sorted) from 3 to 5 mice per condition. Number inside the pie chart indicates total number of sequences analyzed. (E) Experimental scheme for the analysis of CSR events in vitro , inducing Dre recombination (medium supplemented with 4-OHT) at day (d)0 (early time point) or day 4 (late time point) of coculture with 40LB feeder cells (+IL-4), and analyzing at day 4 or day 8, respectively. (F) Left panel: representative flow cytometry histogram showing Foxo1 intracellular expression in Foxo1 WT hom ( Foxo1 wt/wt —blue) and Foxo1 WT het ( Foxo1 fl/wt —brown) iGCBCs at day 4 of coculture. Foxo1 FMO is depicted in gray. Right panel: quantification of the MFI of Foxo1 subtracting the FMO. (G) Percentage of IgG1 + (left) or IgE + (right) iGCBCs at day 4 in Foxo1 WT cells. (H and I) Percentage of ZsGreen + iGCBCs (iGCs; left panel), percentage of IgG1 + and IgE + iGCBCs (middle panel), and Foxo1 MFI in iGCBCs (right panel) at (H) day 4—early time point—or at (I) day 8—late time point—of analysis. (J) Representative flow cytometry histogram showing Foxo1 intracellular expression in EtOH-treated (KO) vs. 4-OHT–treated (rescued) iGCBCs from experimental mice. Statistics: Mann–Whitney test, *P ≤ 0.05; **P ≤ 0.01; ns, not significant. Each dot represents one mouse. Data are from at least two independent experiments. Horizontal lines indicate the median. FMO, fluorescence minus one; MFI, median fluorescence intensity.

Article Snippet: The following antibodies were used: B220-BV785 (clone RA3-6B2; #103246; RRID #AB_2563256; BioLegend), CD19-BV650 (clone 6D5; #115541; RRID #AB_11204087; BioLegend), CD38-AF700 (clone 90; #56-0381-82; RRID #AB_657740; eBioscience), CD86-BV421 (clone GL-1; #105032; RRID #AB_2650895; BioLegend), CD86-PE-Cy7 (clone GL-1; #105014; RRID #439783; BioLegend), CD95/FAS-PE (clone Jo2; #554258; RRID #AB_395330; BD Biosciences), CD95/FAS-PE-Cy7 (clone Jo2; #557653; RRID #AB_396768; BD Biosciences), CXCR4-bio (clone 2B11; #13-9991-82; RRID #AB_10609202; Invitrogen), FOXO1-AF647 (clone C29H4; #72874; RRID #AB_2799829; Cell Signaling), GL7-PE (clone GL7; #144608; RRID #AB_2562926; BioLegend), IgE-BV605 (clone R35-72; #744281; RRID #AB_2742118; BD Biosciences), IgG1-APC (clone A85-1; #560089, RRID #AB_1645625; BD Biosciences), IgG1-BV510 (clone A85-1; #740121; RRID #AB_2739879; BD Biosciences), IgG1-PE (clone A85-1; #550083; RRID #AB_393553; BD Biosciences), IgM-bio (clone Il/41; #13-5790-81; RRID #AB_466675; eBioscience), and Streptavidin-BV605 (#405229; BioLegend).

Techniques: Mutagenesis, Sequencing, Modification, Plasmid Preparation, Homologous Recombination, Control, In Vitro, Flow Cytometry, Expressing, MANN-WHITNEY, Fluorescence

Sequential mutagenesis at the endogenous Foxo1 locus. (A) Schematic representation of in vitro induction of the Foxo1 stopRox allele by Cγ1-CDE (left) and cDNA sequencing in activated Cγ1-CDE, Foxo1 stopRox splenic B cells (right). Expression of the targeted allele is observed only in the presence of 4-OHT, as demonstrated by the detection of the synonymous protospacer adjacent motif (PAM) mutation introduced during the targeting strategy of the transgenic strain. (B) Genetic scheme of recombination events after Cre and Dre activation at the Foxo1 locus of Foxo1 fl/stopRox animals. (C) Experimental scheme (top) and representative gating strategy (bottom) employed for the phenotypic analysis of the GC compartment. (D and E) Top: representative flow cytometry plots measuring the percentage of DZ and LZ (D) and IgG1 + (E) splenic GCBCs of mice of the indicated genotypes at day (d)14 after immunization. Bottom: quantification of the DZ/LZ ratio (D) and the percentage of IgG1 + (E) ZsGreen − (gray) and ZsGreen + (green) splenic GCBCs. Statistics: Mann–Whitney test, **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Each symbol represents one mouse. Data are from three independent experiments. Horizontal lines indicate the median.

Journal: The Journal of Experimental Medicine

Article Title: FOXO1 re-expression with a dual-recombinase allele rescues class switching in germinal center B cells

doi: 10.1084/jem.20241136

Figure Lengend Snippet: Sequential mutagenesis at the endogenous Foxo1 locus. (A) Schematic representation of in vitro induction of the Foxo1 stopRox allele by Cγ1-CDE (left) and cDNA sequencing in activated Cγ1-CDE, Foxo1 stopRox splenic B cells (right). Expression of the targeted allele is observed only in the presence of 4-OHT, as demonstrated by the detection of the synonymous protospacer adjacent motif (PAM) mutation introduced during the targeting strategy of the transgenic strain. (B) Genetic scheme of recombination events after Cre and Dre activation at the Foxo1 locus of Foxo1 fl/stopRox animals. (C) Experimental scheme (top) and representative gating strategy (bottom) employed for the phenotypic analysis of the GC compartment. (D and E) Top: representative flow cytometry plots measuring the percentage of DZ and LZ (D) and IgG1 + (E) splenic GCBCs of mice of the indicated genotypes at day (d)14 after immunization. Bottom: quantification of the DZ/LZ ratio (D) and the percentage of IgG1 + (E) ZsGreen − (gray) and ZsGreen + (green) splenic GCBCs. Statistics: Mann–Whitney test, **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001; ns, not significant. Each symbol represents one mouse. Data are from three independent experiments. Horizontal lines indicate the median.

Techniques: Mutagenesis, In Vitro, Sequencing, Expressing, Transgenic Assay, Activation Assay, Flow Cytometry, MANN-WHITNEY

Re-expression of FOXO1 at the peak of the GC reaction fully restores the FOXO1-associated transcriptional program in ZsGreen + rescued GCBCs. (A–E) ZsGreen + and ZsGreen − GCBCs from experimental and control mice were sorted at day (d)14 after immunization (TAM d10–12) to perform RNA sequencing. (A) Experimental scheme (left) and overview of mouse genotypes (right, n = 2–3 mice per genotype). (B) PCA from the 500 most varying genes. Each dot represents one sample. (C) Heatmap showing Pearson’s correlation coefficients between log 2 fold changes in gene expression profiles. (D and E) GSEA using published up- and downregulated Foxo1 (D) and DZ (E) gene signatures ( ; ). Statistics: CERNO test. ES, enrichment score.

Journal: The Journal of Experimental Medicine

Article Title: FOXO1 re-expression with a dual-recombinase allele rescues class switching in germinal center B cells

doi: 10.1084/jem.20241136

Figure Lengend Snippet: Re-expression of FOXO1 at the peak of the GC reaction fully restores the FOXO1-associated transcriptional program in ZsGreen + rescued GCBCs. (A–E) ZsGreen + and ZsGreen − GCBCs from experimental and control mice were sorted at day (d)14 after immunization (TAM d10–12) to perform RNA sequencing. (A) Experimental scheme (left) and overview of mouse genotypes (right, n = 2–3 mice per genotype). (B) PCA from the 500 most varying genes. Each dot represents one sample. (C) Heatmap showing Pearson’s correlation coefficients between log 2 fold changes in gene expression profiles. (D and E) GSEA using published up- and downregulated Foxo1 (D) and DZ (E) gene signatures ( ; ). Statistics: CERNO test. ES, enrichment score.

Techniques: Expressing, Control, RNA Sequencing, Gene Expression

Journal: The Journal of Experimental Medicine

Article Title: FOXO1 re-expression with a dual-recombinase allele rescues class switching in germinal center B cells

doi: 10.1084/jem.20241136

Figure Lengend Snippet: Transcriptional profile of ZsGreen − (not-rescued) GCBCs resembles the FOXO1-KO transcriptional program. (A and B) GSEA using published up- and downregulated Foxo1 (A) and DZ (B) gene signatures ( ; ). Statistics: CERNO test. n = 2–3 mice per genotype. ES, enrichment score.

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