Figure 4. cGAS is required for the innate immune response to DNA in HaCaT keratinocytes.

(a) Immunoblot analysis of wild-type (WT) and cGAS −/− HaCaT cells, mock transfected or transfected with 1 μg ml⁻¹ HT DNA for 6 h. (b–e) qRT-PCR analysis of cGAS +/+ and cGAS −/− HaCaT cells that were mock transfected or transfected with 1 μg ml⁻¹ HT DNA for 6 h. mRNA levels were normalized to β-actin mRNA levels and mock transfections. IFNβ (b), CCL5 (c), ISG56 (d) and IL6 (e) mRNA levels are shown. (f) qRT-PCR analysis CCL5 mRNA from cGAS +/+ and cGAS −/− HaCaT cells infected with MVA (MOI=5) for 6 h. (g) HEK293T cells were transfected with a firefly luciferase reporter construct under the control of the IFN-β promoter, a Renilla luciferase transfection control, 10 ng STING-Flag plasmid, 1 ng cGAS-Flag and 35 or 70 ng HA-IFI16 expression plasmids, as indicated. Firefly luciferase activity was measured 24 h post transfection, and normalized to Renilla luciferase activity. (h) HEK293T cells were transfected with a firefly luciferase reporter construct under the control of the IFNβ promoter, Renilla luciferase transfection control and 10 ng STING-Flag expression plasmid. In addition, 1 or 5 ng cGAS or TRIF expression constructs were co-expressed with 35 ng HA-IFI16 plasmid or empty vector, as indicated. Relative Firefly luciferase activity was quantified 24 h post transfection. Data are representative of at least three independent experiments, and presented as mean values of triplicate samples. Error bars indicate s.d. *P<0.05, **P<0.01, ***P<0.001 Student's t-test.