Supplementary Figure 4: FAM35A and C20orf196 directly affect class switch recombination.
From: Shieldin complex promotes DNA end-joining and counters homologous recombination in BRCA1-null cells
a, Clonogenic survival assay following IR treatment using wild-type, Fam35ako or C20orf196ko mouse ES cells (right panel shows AUC). Bars represent means ± SEM, one-way Anova; n=3 independent experiments, with individual data points plotted over bars. b, Genotypes of CH12-Cas9 cell knockout clones used in CSR assays confirmed by Topo-cloning and Sanger sequencing. c, Flow cytometry profiles showing the percentage of IgA+ cells for indicated CH12-Cas9 cell clones (genotypes) after 3 days stimulation with anti-CD40, IL-4 and TGF-β. Cell clone numbers are indicated; representative of 3 independent experiments. d, CSR assay in C20orf196ko cells complemented with C20orf196. Bars represent means ± SEM, one-way Anova; n=3 independent experiments, with individual data points plotted over bars. e, CH12-Cas9 clones were plated at 50,000 cells/ml and counted after 3 days stimulation with anti-CD40, IL4, and TGF-β. Bars represent means ± SEM, one-way Anova; n=3 independent experiments, with individual data points plotted over bars. For a, d and e, *p<0.05, ***p<0.001, ****p<0.0001, ns=not significant (p≥0.05); statistical source data including the precise p values can be found in Supplementary Table 5. f, Igh, α germ-line transcripts (αGLT) and Aid mRNA were quantified by semi-quantitative RT–PCR using 2.5-fold serial dilutions of cDNA made from CH12-Cas9 cells and indicated CH12-Cas9 knockout cell clones after 2 days stimulation with anti-CD40, IL4, and TGF-β. Hprt was used as a control for transcript expression. Immunoblots are representative of two independent experiments with unprocessed scans of immunoblots in Supplementary Fig 8.
