Fig. 5: Loss of XRCC4 attenuates RIG-I immune signaling.

a A549 cells were transfected with 3p-hpRNA (0.5 μg/ml, 8 h). The cells were lysed, and cytosolic fractions were immunoprecipitated with anti-RIG-I antibody. The beads were treated with RNase A, boiled and blotted with indicated antibodies. b A549 cells were transfected with 3p-hpRNA (0.5 μg/ml) for the indicated time. Cells were subjected to subcellular fractionation into soluble cytoplasmic (SC), cytoplasmic membranous (CM), and soluble nuclear (SN) components. Western blot was performed with indicated antibodies. c A549 cells were transfected with 3p-hpRNA (0.5 μg/ml) for the indicated time. Cells were subjected to fractionation into cytosolic (Cyto) and mitochondria (Mito) components. Western blot was performed with indicated antibodies. d IFN-β RNA levels in control and XRCC4 knockdown A549 cells transfected with 3p-hpRNA (0.5 μg/ml, 12 h) were analyzed by qRT-PCR. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test. e Control and XRCC4 knockdown A549 cells expressing RIG-I shRNA were transfected with 3p-hpRNA (0.5 μg/ml, 12 h). IFN-β RNA levels were detected by qRT-PCR. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test. f XRCC4 knockdown HEK293T cells re-expressing wild type (WT) or XRCC4 mutant lack of the head and coil-coiled domains (ΔHD/CCD) were transfected with Flag- and GFP-tagged RIG-I and then 3p-hpRNA (0.5 μg/ml, 8 h). The cells were lysed, and immunoprecipitated with anti-Flag agarose beads. The beads were boiled and blotted with indicated antibodies. g Control and XRCC4 knockdown HEK293T cells were transfected with Flag-RIG-I and His-Ub and then 3p-hpRNA (0.5 μg/ml, 8 h). Cell lysates were immunoprecipitated with Ni-NTA (His) beads, and then blots were probed with indicated antibodies. h XRCC4 knockdown cells re-expressing wild type (WT) or XRCC4 mutant (ΔHD/CCD) were transfected with Flag-RIG-I and then 3p-hpRNA (0.5 μg/ml, 8 h). The cells were lysed, and immunoprecipitated with anti-Flag agarose beads. The beads were boiled and blotted with indicated antibodies. i XRCC4 knockdown cells re-expressing WT or XRCC4 mutant (ΔHD/CCD) were transfected with 3p-hpRNA (0.5 μg/ml, 12 h). IFN-β RNA levels were detected by qPCR. Data are presented as mean values ± SEM from three independent experiments. P values are determined by unpaired two-sided t-test.