Fig. 4: Endogenous host RNA produced during MIRI activate and initiate the RIG-I cascade.

a Co-immunoprecipitation assays were performed to detect lysine 63 residues (K63)-type ubiquitination of RIG-I in vivo at 6 h after reperfusion or sham. The RIG-I protein was used as the bait protein to capture the interaction protein candidates, and immunoprecipitation (IP) and input represent the protein sample to be detected, respectively. b Statistical data of protein expression in (a). c K63-type ubiquitination of MAVS in vivo was determined by co-immunoprecipitation assay. The MAVS protein acts as the bait protein to capture the interaction proteins, and IP and input represent the protein sample to be detected, respectively. d Statistical data of protein expression in (c). e RIG-I and MAVS expression was determined by western blot in HEK293T cells transfected with RNA extracted from heart tissues at remote area, area at risk and infarct area after 6 h post-reperfusion or sham group, and RNA extracted from unrelated normal cells was negative control RNA (NC-RNA). f Statistical data of protein expression in (e). g Transcriptional levels of transforming growth factor-β-activated kinase 1 (TAK1), tumor necrosis factor-associated factor family 6 (TRAF6), interferon-α (IFN-α) and interferon-γ (IFN-γ) in HEK293T cells treated as described in (e). h K63-type ubiquitination of RIG-I was determined by co-immunoprecipitation at 48 h after transfection with total RNA from heart tissues at 6 h after reperfusion versus controls. i Statistical data of protein expression in (h). j Immunofluorescence staining of double-stranded RNA (dsRNA) in myocardial tissue sections in WT mice at 1 day after reperfusion and in sham group. α-actin indicates cardiomyocytes. Scale bar, 20 μm. b, d, f, g, i Data are shown with 3 biologically independent samples. Data are means ± standard deviation; ***p < 0.001, ****p < 0.0001 by one-way analysis of variance statistics with Tukey’s multiple comparisons test (g) and *p < 0.05, **p < 0.01, ***p < 0.001, **** p < 0.0001 by two-way analysis of variance with Tukey’s multiple comparisons test (f, i); ****p < 0.0001 by unpaired 2-tailed Student t test (b, d); ns, p ≥ 0.05. Source data are provided as a Source Data file.