Extended Data Fig. 8: Developing RIG-I interfering peptides (RIPs) to prevent Cys864-Cys869 disulfide linkage can disrupt RIG-I LLPS thereby reducing RIG-I-mediated autoimmune responses.
From: Targeting a key disulfide linkage to regulate RIG-I condensation and cytosolic RNA-sensing
a, Schematics show the C268F and E373A mutations within the RIG-I protein (left). Coomassie blue staining of GFP-RIG-I proteins (right). b, Representative images and quantification of the in vitro droplets formed by indicated protein; n = 3; P = 0.0022 (C268F vs. WT), P = 0.0078 (E373A vs. WT). c, SDD–AGE analysis of RIG-I aggregation and immunoblot (IB) analysis of RIG-I KO cells transfected with indicated plasmids. d, Immunofluorescence microscopy and DAPI staining of HeLa cells transfected with indicated plasmids. Quantified percentage of cells with RIG-I puncta was shown; n = 3. e, Normalized IFNB1 mRNA expression and IB analysis (right) of RIG-I KO HEK293T cells transfected with indicated plasmids; n = 3; ***P = 0.0002. f, Normalized CXCL10 and ISG56 mRNA expression in RIG-I KO HEK293T cells transfected with the indicated plasmids; n = 3. g, Related to Fig. 8d, normalized CXCL10 and ISG56 mRNA expression was shown; n = 3. h, Droplet formation (left) and quantification (right) of GFP-RIG (10 μM) treated with control BSA or RIPs (5 µM) for 30 min. n = 3. i, Related to Fig. 8h, normalized CXCL10 (left) and ISG56 (right) mRNA expression was shown; n = 3. j, Representative images (left) and quantification (right) of the in vitro droplet formation by GFP-RIG-I WT, C864S or C869S (10 μM) and mixed with control PBS or mtRNA (1 μg); n = 3. k, Related to Fig. 8i, normalized CXCL10 and ISG56 mRNA expression was shown; n = 3. l, IB analysis of p-IRF3, p-TBK1 and IRF3 dimerization and total IRF3, TBK1 in Rig-IWT/WT and Rig-IC865S/C865S MEFs transfected without (−) or with (+) mtRNA (2.5 μg/mL) for 12 h; n = 3. m, Related to Fig. 8l, normalized Cxcl10 and Ccl5 mRNA expression was shown; n = 3; ***P = 0.0001. Data are representative of at least three independent experiments and shown as mean ± s.d. Scale bar, 10 μm (d), 20 μm (b,h,j). Statistical analysis was performed using a two-tailed Student’s t-test (b,d–k,m); ****P < 0.0001; ns, not significant. Exact P values, source numerical data and unprocessed blots are provided.
