Figure 6: IFI16 is required for cGAMP-induced STING activation.

(a) IFI16 +/+ and IFI16 −/− HaCaT cells were transfected with 20 μg ml⁻¹ synthetic cGAMP, and CCL5 mRNA induction was analysed by qRT-PCR at the time points indicated, and normalized to β-actin mRNA. (b) IFI16 +/+ and IFI16 −/− HaCaT cells were infused with 15 μM cGAMP by digitonin-mediated permeabilization, and CCL5 protein in supernatants was quantified by ELISA 24 h post stimulation. (c) HaCaT cells were permeabilized with digitonin and infused with 15 μM cGAMP for 2, 4 or 6 h. Phosphorylation of STING, of IRF3 at Ser396 (pIRF3) and TBK1 at Ser172 (pTBK1) was analysed by SDS–PAGE and western blotting. (d) HaCaT cells were transfected with 20 μg ml⁻¹ cGAMP for 4 h, and the translocation of endogenous IRF3 was observed by confocal microscopy. Cells were stained for IRF3 (green), IFI16 (red) and DNA (DAPI, blue). Scale bar, 20 μm. (e) Cells as in d were scored for predominantly cytosolic (C), predominantly nuclear (N) and evenly distributed nuclear and cytosolic (N+C) localization of IRF3. At least 200 cells were counted per sample. (f) Schematic representation of the co-culture of HaCaT cells with cGAS-expressing HEK293T cells. Endogenously produced cGAMP can diffuse through gap junctions from the cGAS-expressing producer HEK293T cells to HaCaT cells, where it can bind STING to induce an innate immune response. (g,h) HEK293T cells were transiently transfected with a cGAS-FLAG expression construct or empty vector (EV) for 6 h, then co-cultured with IFI16 +/+ or IFI16 −/− HaCaT cells for 18 h. (g) Immunoblot analysis of cGAS-FLAG, IFI16 and STING protein expression in the co-culture. (h) qRT-PCR analysis of CCL5 mRNA expression in IFI16 +/+ or IFI16 −/− HaCaT cells grown in monoculture (−) or co-cultured with HEK293T cells expressing cGAS-FLAG or empty vector (EV). Data show means of triplicate samples with s.d. Shown are representatives of at least two independent experiments each in two IFI16 −/− cell clones.