Figure 1. IFI16 is required for DNA but not RNA sensing in HaCaT keratinocytes.

(a) Immunoblot analysis of wild-type (IFI16 +/+) HaCaT and two IFI16 −/− HaCaT clones. (b–i) Quantitative real-time PCR (qRT-PCR) analysis of mRNA expression levels normalized to β-actin mRNA and mock transfection in IFI16 +/+ and IFI16 −/− HaCaT cells, as indicated. (b) qRT-PCR analysis of IFN-β mRNA expression in IFI16+/+ and two IFI16 −/− HaCaT cells clones transfected with 1 μg ml⁻¹ HT DNA for the times indicated. (c) qRT-PCR analysis of IFN-β mRNA 6 h post transfection with 1 μg ml⁻¹ of a 70nt dsDNA oliogonucleotide (70mer) or circular pcDNA3.1 plasmid. (d) IFN-β mRNA induction 6 h after transfection with 1, 10 or 100 ng ml⁻¹ poly(I:C). (e) Time course of ISG56 mRNA expression following transfection with 1 μg ml⁻¹ HT DNA. (f) ISG56 mRNA expression 6 h post transfection with 1 μg ml⁻¹ 70mer oligonucleotide or 100 ng ml⁻¹ poly(I:C). (g) qRT-PCR analysis of CCL5 mRNA expression following transfection with 1 μg ml⁻¹ HT DNA for the times indicated. (h) Relative CCL5 mRNA expression levels 6 h post transfection with 1 μg ml⁻¹ 70mer oligonucleotide or 100 ng ml⁻¹ poly(I:C). (i) CCL5 mRNA expression levels 6 h post transfection with 1 μg ml⁻¹ of Y-G3 or Y-C3 oligonucleotides. (j) Secreted CCL5 (Rantes) protein detected by ELISA in the supernatants of IFI16 +/+ or IFI16 −/− HaCaT cells transfected with 1 μg ml⁻¹ HT DNA, Y-G3 or Y-C3 DNA for 24 h. (k) ELISA quantitation of CCL5 protein in supernatants from IFI16 +/+ and IFI16 −/− HaCaT cells stimulated with 5 μg ml⁻¹ extracellular (EC) poly(I:C) added to the medium for 24 h. (l) ELISA quantitation of CXCL10 (IP-10) protein in supernatants of IFI16 +/+ or IFI16 −/− HaCaT cells transfected with 1 μg ml⁻¹ 70mer oligonucleotide or HT DNA. All qRT-PCR and ELISA data are presented as mean values of biological triplicates. Error bars indicate s.d. *P<0.05, **P<0.01, ***P<0.001 Student's t-test. Data are representative of at least two experiments in two independent IFI16-deficient cell clones.