Extended Data Fig. 4: STING condensation requires STING intact transmembrane topology and STING dimerization.
From: The STING phase-separator suppresses innate immune signalling
a, c, d, e, f, Diagram of STING transmembrane domains69 (a, top left). 293T cells were transfected with plasmid expressing STING-GFP or indicated STING truncations (a, c, d, f,). The WT, TBK1−⁄− or IRF3−⁄− HeLa cells were transfected with plasmid expressing equal amounts of STING-GFP for 18 h (e). Protein expression of the indicated STING truncation was analyzed by western blot using antibodies against GFP and GAPDH (a, c, d, f,). Representative confocal images of STING condensates were shown (a, c, d, e, f,). MOD (a) were 32.5 (WT), 31.9 (dTM1), 33.7 (d35-44), 26.5 (dTM2), 35.6 (dTM3), 25.2 (d107-116), 24.9 (dTM4), 31.5 (d107-134). MOD (d) were 44.5 (WT), 33.3 (C88S), 37.3 (C91S), 32.9 (C88S/C91S, CC/SS). MOD (e) were 65.9 (WT), 73.7 (TBK1−⁄−), 66.5 (IRF3−⁄−). MOD (f) of representative images were 94.5 (WT) and 74.4 (d371-376). Area and integrated optical density (IOD) of STING condensates per cell were quantified. 25 fields were examined and stitched for quantification as one repeat. (mean ± SD, n = 2 repeats; one-way ANOVA (a, c, d); p < 0.0001 (a, area); p = 0.0023 (dTM1), p = 0.0005 (d35-44), p = 0.0009 (dTM2), p = 0.0076 (dTM3), p = 0.0044 (d107-116), p = 0.0012 (dTM4), p = 0.0007 (d107-134) (a, IOD); p < 0.0001 (c); p > 0.05 (d, e)). b, 293T cells were transfected with plasmid expressing cGAS, STING WT or indicated mutants with an IFNβ–Luc reporter system for 24 h before a luciferase assay was performed (mean ± SD, n = 2 biologically independent samples; one-way ANOVA, p < 0.0001). For all Extended Data Fig. 4, data were representatives of at least three independent experiments. Confocal images are representatives of at least three independent experiments in which >95% of the cells displayed similar pattern. Scale bar, as indicated. Nuc, Nucleus. See uncropped gel images and numerical data in source data.
