Figure 3. cGAS-produced cGAMP(2′-5′) passes through gap junctions to trigger STING activation in bystander cells.

a, Confocal microscopy of HEK cells and HEK cGAS* cells loaded with calcein and added to HEK STING cells for 4 h. b, Co-culturing was performed as in a and after 0–8 h HEK STING cells were analysed by fluorescence microscopy for STING aggregation in nine independent visual fields. A dot-blot diagram correlating calcein-positive HEK STING cells with STING aggregate formation is presented (y = 1.27x, R² = 0.85). AU, arbitrary units. c, HEK STING cells were co-cultured with HEK cGAS* in the presence of CBX as indicated for 4 h and studied for STING activation in nine independent visual fields. Data are presented as mean and s.e.m. d, IRF3 phosphorylation in HEK STING cells with HEK cells, with HEK cGAS* cells or with CMA in the presence or absence of CBX (100 μM, 150 μM and 200 μM) (3 h). e, The scrape loading technique. f, Fluorescence images of wounded HEK STING cells incubated with nothing, cGAMP(2′-5′) or cGAMP(2′-5′) with 150 μM CBX. HEK STING cells with CMA served as control (dashed line, scratch margins; arrows, STING complexes). One representative experiment out of two independent experiments is shown (a-d, f). *P < 0.05, ***P < 0.001.