Figure 7: IFI16 acts on STING to promote its activation by cyclic di-nucleotides.
From: IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes
(a) IFI16 +/+ or IFI16 −/− HaCaT cells were permeabilized with digitonin and infused with 15 μM cGAMP or its non-hydrolysable analogue cGAM(PS)2 for 6 h. CCL5 mRNA expression was analysed by qRT-PCR. (b) Cells were permeabilized and infused with 15 μM cGAMP or cGAM(PS)2 for 4 h, and lysates were analysed by western blotting for phosphorylation of STING, TBK1 at Ser172 (pTBK1) and IRF3 at Ser396 (pIRF3). (c) Cells were transfected with 100 μg ml−1 cyclic di-AMP for 6 h, and IFN-β mRNA levels were quantified by qRT-PCR. (d) STING was immunoprecipitated from HaCaT cells transfected with 5 μg ml−1 HT DNA for the times indicated. Lysates and immunoprecipitates (IP) were analysed by SDS–PAGE and western blotting. (e) HEK293T cells were transfected with a firefly luciferase reporter construct under the control of the IFNβ promoter, a Renilla luciferase transfection control, 2 ng STING-FLAG plasmid and 150 ng empty vector (EV) or IFI16 expression constructs as indicated: full-length IFI16 (fl), the IFI16 HINb domain (HINb), or the IFI16 pyrin domain (PYD). Firefly luciferase activity was measured 24 h post transfection, and normalized to Renilla luciferase activity. Data are representative of at least two independent experiments. qRT-PCR and luciferase data are expressed as means of triplicate samples; error bars represent s.d. *P<0.05, **P<0.01, ***P<0.001 Student’s t-test.
