Fig 2: LGP2 is required for the induction of a type I IFN response upon siRNA-mediated depletion of ADAR1 in HEK293, related to Fig 1 PMA-differentiated THP-1 WT, RIG-I-, MDA5-, and LGP2-knockout cells were transfected with siCtrl (siC) or siADAR1 (siA). Protein lysates were prepared 56 h post-transfection and ADAR1 knockdown efficiency was monitored by immunoblot analysis. Data correspond to the biological replicate shown in Fig 1B.Expression level and type I IFN inducibility of relevant proteins in HEK293 and THP-1. HEK293 and PMA-differentiated THP-1 cells were treated with or without recombinant type I IFN. Protein lysates were analyzed by SDS-PAGE followed by immunoblotting with the indicated antibodies (n = 3). *, nonspecific band.Kinetics of siRNA-mediated depletion of ADAR1 and induction of the type I IFN response in HEK293. HEK293 cells stably expressing FLAG-LGP2 were transfected with an siRNA targeting ADAR1 (siADAR1) and harvested at the indicated time points post-transfection. ADAR1 knockdown and IFIT1 upregulation were monitored by RT-qPCR analysis (using Taqman probes) and normalized to ACTB. Data are means ± s.d. from one experiment.HEK293 WT cells were transfected with siADAR1 or a control siRNA (siCtrl) and 8 h later with increasing amounts of a vector encoding FLAG-LGP2. As a control, cells were transfected with 250 ng of an empty vector (EV). Cells were harvested 80 h post siRNA transfection. RT-qPCR analysis was used to monitor IFN-ß and IFIT1 expression, ADAR1 knockdown, and LGP2 (DHX58) expression. All transcripts were normalized to ACTB. Data are means ± s.d. from a representative of two biological replicate experiments.Cells were treated as in (D). Protein lysates were prepared and analyzed by SDS-PAGE followed by immunoblotting using the indicated antibodies. Source data are available online for this figure.

Fig 3: 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-ß1, 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 ?H2AX 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 ?H2AX 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.

Fig 4: Alterations of RIG-I partially abolish lncRNA Ftx/miR-545-mediated HCC cell viability, colony formation, proliferation and cell cycle progressionA,F. LncRNA Ftx/miR-545-suppressive Hep3B cells that were transfected with EV or RIG-I expression plasmid and lncRNA Ftx/miR-545-overexpressing SMMC-7721 that were transfected with control siRNA or RIG-I siRNA were subjected to western blot analysis for RIG-I. n = three repeats with similar results. The correlations between lncRNA Ftx/miR-545 effects and RIG-I knockdown or overexpression are shown in the B,G. cell viability, C,H. colony formation, D,I. cell proliferation and E,J. cell cycle progression. RIG-I knockdown abrogated the effects of lncRNA Ftx/miR-545 knockdown on Hep3B cells. RIG-I overexpression induced effects that were opposite to those stimulated by pcDNA-Ftx or miR-545. n = three independent experiments. *P< 0.05, **P< 0.01.

Fig 5: CypA inhibits TRIM25-mediated K48-linked ubiquitination of MAVS.(A) Luciferase activity of lysates in 293T/CypA- cells transfected for 24 hr with IFN-ß-Luc and Flag-TRIM25, Flag-Smurf1, Flag-AIP4, Flag-RNF125, or Flag-RNF5, along with CypA or an empty vector and then treated with SeV for 6 hr. Results are presented relative to the luciferase activity in control cells transfected with luciferase reporter and empty vector. (B) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids for 24 hr. (C) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids for 24 hr, followed by immunoprecipitation with anti-MAVS antibody. (D) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids for 24 hr, followed by immunoprecipitation with anti-Flag beads. (E) Immunoblot analysis of lysates in WT and Ppia-/- BMDMs infected with SeV for 6 hr, followed by immunoprecipitation with control mouse IgG or anti-MAVS antibodies. Lysates and immunoprecipitation extracts were probed with K48-Ub, MAVS and CypA antibodies. (F) Immunoblot analysis of lysates in 293T/CypA- cells transfected with various combinations of plasmids, followed by immunoprecipitation with anti-Flag beads. (G and H) Immunoblot analysis of lysates in 293T/CypA+ cells transfected for 24 hr with Flag-CypA (G) or Flag-TRIM25 (H), along with HA-tagged deletion constructs of MAVS, followed by immunoprecipitation with anti-Flag beads. (I) Immunoblot analysis of lysates in 293T/CypA+ cells transfected with HA-Ub, plus Myc-MAVS, Myc-MAVS KK-AA (K371A plus K420A), or the double point substitution construct Myc-MAVS KK-RR (K7R plus K10R), along with Flag-TRIM25 or an empty vector, followed by immunoprecipitation with anti-Myc beads. (J) Quantitative PCR analysis of IFNB1 mRNA in 293T/RIG-I-/- cells pretreated for 1 hr with BAY 11–7082 (5 µM) or DMSO, and then transfected for 48 hr with Flag-RIG-I, Flag-MAVS or an empty vector, along with scrambled siRNA or CypA siRNA (top). The phosphorylated IRF3 and p65 were detected by immunoblot (below). (K) Quantitative PCR analysis of SeV M mRNA in 293T/RIG-I-/- cells pretreated for 1 hr with BAY 11–7082 (5 µM) or DMSO, then transfected for 48 hr with Flag-RIG-I, Flag-MAVS or an empty vector, along with scrambled siRNA or CypA siRNA, and then infected with SeV for 6 hr. Results are presented relative to mRNA level of SeV M in control cells transfected with empty vector and infected with SeV. Data are shown as mean ± SD (n = 3). *p<0.05 and **p<0.01 (unpaired, two-tailed Student’s t-test). Data are representative of at least three independent experiments.DOI: http://dx.doi.org/10.7554/eLife.24425.01810.7554/eLife.24425.019Figure 7—source data 1.Quantification of luciferase activity, IFN-ß production and SeV replication for Figure 7.DOI: http://dx.doi.org/10.7554/eLife.24425.019