Extended Data Fig. 3: STING condensation requires STING IDR.

a, Human STING^309-379 (top) and mammalian STING (bottom) C-terminal region (309-379), were intrinsically unstructured and conserved (https://iupred2a.elte.hu/). b, 293T cells were transfected with plasmid expressing STING^WT-GFP (bottom) or STING ^d309-379-GFP (top). After 12 h, cells with similar fluorescence intensity were chosen and confocal images were taken. Only in cells transfected with STING^WT plasmid, STING condensates emerged and grew larger with increased STING protein expression. Percentage of cells with STING condensates in ten fields was quantified (right, means ± SD). c, HeLa TMEM173^−⁄−-STING-GFP or TMEM173^−⁄−cells stably expressing STING^d309-379-GFP were treated with 2’3’-cGAMP for 3 h. Representative confocal images of STING condensates were shown. Number of condensates in each field was counted (right, mean ± SD, n = 10 fields). d, g, Quantification of integrated optical density (IOD) of condensates in Fig. 2e (d), and Fig. 2f (g), (mean ± SD, n = 2 repeats; one-way ANOVA (d), p = 0.001 (d309-379), p = 0.0013 (d309-342); unpaired t test (e), p = 0.0073). ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. 25 fields were examined and stitched for quantification as one repeat. The expression level of indicated proteins was analyzed by western blot using antibodies against GFP and GAPDH. e, STING E³³⁶ and E³³⁷ were conserved among the indicated species. f, STING disorder tendency of STING-WT^309-342 and STING-E³³⁶G/E³³⁷G^309-342 (http://pondr.com/). For all Extended Data Fig. 3, data were representatives of at least three independent experiments. Confocal images represented at least three independent experiments in which >95% of the cells displayed similar pattern. Scale bar, as indicated. See uncropped gel images and numerical data in source data.

Source data