Extended Data Fig. 3: The Cys864-Cys869 intermolecular disulfide bond located in R3 is required for an efficient RIG-I LLPS.
From: Targeting a key disulfide linkage to regulate RIG-I condensation and cytosolic RNA-sensing
a, Representative confocal microscopy images showing the effects of DTT treatments on droplet formation of GFP-RIG-I proteins with or without 2 μM dsRNA incubation; n = 3. b, Representative confocal microscopy images and quantitative data showing the effects of DTT treatments on droplet formation of GFP-SARS2-NP proteins (20 μM); n = 3; P = 0.4874 for DTT vs. DMSO. Right: Coomassie blue staining of purified SARS2-NP. c, Immunofluorescence microscopy and DAPI staining of HeLa cells transfected with GFP-SARS2-NP followed by treatment with DMSO or 0.2 mM DTT for 1 h (left). Quantification of cells with GFP-SARS2-NP puncta was shown (right); n = 3; P = 0.8142 for DTT vs. DMSO. d, Representative images and quantitative data showing the effects of PDI treatments on droplet formation of normal GFP-RIG-I proteins; n = 3; P = 0.5886 for GSH + GSSG vs. untreated, P = 0.9482 for PDI + GSH + GSSG vs. untreated. e, Representative images and quantitative data showing the effects of PDI treatments on droplet formation of reduced GFP-RIG-I proteins (20 μM); n = 3; P = 0.0980 for GSH + GSSG vs. pretreated. f, SDD–AGE analysis of RIG-I aggregation (top) and SDS–PAGE (bottom) of SeV-stimulated RIG-I knockout cells transfected with indicated plasmids. g, Coomassie blue staining of indicated proteins. h, Sedimentation analysis of GFP-RIG-I WT, C864S and C869S proteins after they were incubated with PBS or dsRNA (top). Quantified band intensity were shown (bottom); n = 3; P = 0.5201 for dsRNA vs. PBS in C864S, P = 0.0815 for dsRNA vs. PBS in C869S. i, Immunofluorescence microscopy and DAPI staining of HeLa cells transfected to express GFP-RIG-I WT, C864S or C869S proteins followed by stimulation with SeV for 12 h (left). Quantification of cells with GFP-RIG-I puncta was shown (right); n = 3; P = 0.0023 for C864S vs. WT, P = 0.0027 for C869S vs. WT. Data are representative of at least three independent experiments. Scale bar, 20 μm (a,b,d,e), 10 μm (c,i). Mean ± s.d., statistical analysis was performed using two-tailed Student’s t-test (b–e,h,i); ****P < 0.0001; ns, not significant. Exact P values, source numerical data and unprocessed blots are provided.
