Figure 4. Connexin 43 and 45 mediate cGAMP(2′-5′) transfer in HEK STING cells.

a, Fluorescence microscopy of HEK STING CX43/45^WT and HEK STING CX43/45^DKO cells left untreated, stimulated with CMA or co-cultured with calcein-loaded HEK cGAS* cells (ratio HEK/HEK STING:HEK cGAS* = 1:0.25) after 8 h. b, Phosphorylation of IRF3 in HEK STING CX43/45^WT and HEK STING CX43/45^DKO cells left untreated, co-incubated with HEK cGAS* cells or stimulated with CMA for 4 h (asterisk indicates nonspecific band). c, HEK STING CX43/45^WT and HEK STING CX43/45^DKO cells were co-cultured with HEK cGAS^low cells (ratios from 1:0.5 to 1:0.0312 HEK STING CX43/45^WT or HEK STING CX43/45^DKO cells:cGAS^low) transfected with pIFN-β-GLuc and luciferase activity was assessed after 20 h. Mean and s.e.m. (biological duplicates) of one representative experiment out of three independent experiments is shown. d, Fluorescence microscopy of HEK STING CX43/45^DKO cells co-cultured with HEK cGAS* cells and stimulated with CMA, transfected with empty vector (pCI) or an expression vector for murine CX45 (mmCX45, 20 h). e, Co-cultures from d were analysed for phosphorylation of IRF3. One representative experiment out of two independent experiments is shown (a, b, d, e). *P < 0.05, **P < 0.01.