Figure S2.
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 Foxo1fl/wt and experimental Foxo1fl/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 Foxo1fl/wt control, Foxo1fl/stopRox experimental, and Foxo1fl/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 (Foxo1wt/wt—blue) and Foxo1 WT het (Foxo1fl/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.

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 Foxo1fl/wt and experimental Foxo1fl/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 Foxo1fl/wt control, Foxo1fl/stopRox experimental, and Foxo1fl/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 (Foxo1wt/wt—blue) and Foxo1 WT het (Foxo1fl/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.

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