Figure 3: IFI16 is required for the DNA-induced activation of STING and IRF3.

(a) Confocal analysis of IFI16 +/+ and IFI16 −/− HaCaT cells that were mock transfected or transfected for 1 h with 5 μg ml⁻¹ HT DNA. Cells were stained for endogenous IFI16 (red) and STING (green). DNA is visualized with DAPI (blue). (b) Cells as in (a) were observed by confocal microscopy and scored for STING clustering. At least 200 cells were counted per sample. (c) Confocal analysis of IFI16 −/− HaCaT cells reconstituted for 6 h with 1 μg ml⁻¹ in vitro transcribed, capped and polyadenylated mRNA encoding GFP or IFI16, followed by transfection with 5 μg ml⁻¹ HT DNA for 1 h. Cells were stained for STING (red), and DNA (DAPI, blue). GFP or AlexaFluor488-stained IFI16 are shown in green. (d) Cells as in c were scored for STING clustering, with at least 300 cells counted per sample. (e) Immunoblot analysis of HaCaT cells treated with 1 μg ml⁻¹ HT DNA for 4 h, and probed for IFI16, STING and β-actin protein levels by Western blotting. (f) HaCaT cells were stimulated with 1 μg ml⁻¹ HT DNA for 6 h or left untreated (UT). STING immunoprecipitates (IP) were treated with λ phosphatase where indicated, and analysed by western blotting. (g) Western blot analysis of IRF3 phosphorylation at Ser396 (pIRF3) and TBK1 phosphorylation at Ser172 (pTBK1) in HaCaT cells transfected with 1 μg ml⁻¹ HT DNA for the times indicated. (h) HaCaT cells were transfected with 5 μg ml⁻¹ HT DNA for 4 h, and the translocation of endogenous IRF3 was analysed by confocal microscopy. Cells were stained for IRF3 (green) and IFI16 (red), DNA is visualized with DAPI (blue). (i) Cells as in (h) were scored for predominately cytosolic (C), predominantly nuclear (N) and evenly distributed nuclear and cytosolic (N+C) localization of IRF3. At least 200 cells were counted per sample. Results are representative of at least two experiments each in two independent IFI16 −/− cell clones. Scale bars, 20 μm.