Fig. 1: RIG-I is recruited to DNA DSB sites and suppresses non-homologous end-joining.

a A549 cells were treated with irradiation (IR, 10 Gy, 2 h). RIG-I protein levels in the cytosolic (C) and nuclear (N) fractions were detected by Western blot. b A549 cells were treated with IR (10 Gy) for the indicated times. RIG-I protein levels in the soluble and chromatin fractions were examined by Western blot. c ER-AsiSI U2OS cells were transfected with empty vector or Flag-RIG-I, and then treated with 4-OHT to induce DSBs. Flag-RIG-I 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. d U2OS-FokI cells were treated with 1 mM Shield-1 and 1 mM 4-OHT for 5 h to induce site-specific double-strand breaks by FokI, and then fixed for immunofluorescence assay. RIG-I (green) localizes to the DSB site in U2OS-FokI cells where the DSB is induced by FokI (red). Scale bar, 10 μm. e Control and RIG-I 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. f Control and RIG-I overexpressing HEK293T cells were transfected with linearized pEYFP plasmid for 12 h, followed by qPCR to detect the ligated EYFP region, normalized to an uncut flanking DNA sequence. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test. g Control and RIG-I knockdown 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. h Control and RIG-I knockdown HEK293T cells were transfected with linearized pEYFP plasmid for 12 h, followed by qPCR to detect the ligated EYFP region, normalized to an uncut flanking DNA sequence. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test. i Control and RIG-I knockdown CH12F3-2a cells were stimulated with ligands (TGF-β1, IL-4, and CD40 ligand), and class switch from IgM to IgA was analyzed. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test. Representative pictures (j) and quantification (k) of γH2AX foci in control and RIG-I knockdown U2OS cells treated with IR (2 Gy) for the indicated time. Data are representative of three independent experiments. Each dot represents a single cell, and 100 cells were counted in each group for this experiment. Error bars represent ±SEM from this experiment. P values are determined by unpaired two-sided t-test. Scale bar, 10 μm. l Quantification of γH2AX foci in control and RIG-I overexpressing U2OS cells treated with IR (2 Gy) for the indicated time. Data are representative of three independent experiments. Each dot represents a single cell, and 100 cells were counted in each group for this experiment. Error bars represent ±SEM from this experiment. P values are determined by unpaired two-sided t-test.