Fig. 3: RIG-I suppresses non-homologous end-joining by disrupting the formation of XRCC4/LIG4/XLF complex at DSB sites.

a HEK293T cells were transfected with Flag-XRCC4 and GFP-RIG-I, and then treated with IR (10 Gy, 1 h). The cells were lysed and immunoprecipitated with anti-Flag agarose beads. The beads were boiled, subjected to SDS-PAGE, and analyzed with indicated antibodies. b HEK293T cells were transfected with Flag-RIG-I, and then treated with IR (10 Gy, 1 h). The soluble and chromatin fractions were separated, subjected to SDS-PAGE, and analyzed with indicated antibodies. c–e ER-AsiSI U2OS cells were transfected with Flag-XRCC4, Flag-XLF, or Flag-LIG4, and then treated with 4-OHT to induce DSBs. Flag-XRCC4 (c), Flag-XLF (d), and Flag-LIG4 (e) accumulation at DNA damage sites generated by AsiSI was detected by ChIP-qPCR. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test. f Schematic representation of XRCC4 constructs used in this study (top). HEK293T cells were transfected with indicated GFP-XRCC4 constructs and Flag-RIG-I. Cell lysates were incubated with anti-GFP agarose beads. The immunoprecipitates were blotted with indicated antibodies (bottom). g Schematic representation of RIG-I constructs used in this study (top). HEK293T cells were transfected with indicated Flag-RIG-I constructs and GFP-XRCC4. Cell lysates were incubated with anti-Flag agarose beads. The immunoprecipitates were blotted with indicated antibodies (bottom). h HEK293T cells were transfected with Flag-XRCC4 and wild type (WT) or RIG-I mutant lack of C-terminal domain (ΔCTD), and then treated with IR (10 Gy, 1–2 h). The cells were lysed and immunoprecipitated with anti-Flag agarose beads. The beads were boiled, subjected to SDS-PAGE, and analyzed with indicated antibodies. i Control and WT or RIG-I mutant (ΔCTD) overexpressing HEK293T cells were transfected with NHEJ reporter, and then cells were harvested for the NHEJ assay. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test.