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. 2017 Feb 13;8:14392. doi: 10.1038/ncomms14392

Figure 2. IFI16 is required for the innate immune response to DNA viruses.

Figure 2

(a) Confocal imaging of HaCaT cells infected with VACV-A3-RFP (MOI=0.1) for 24 h and stained with FITC-labelled IFI16 antibody (green). A3-RFP is shown in red, DNA is stained with DAPI (blue). (b) Confocal imaging of HaCaT cells infected with HSV-1 (MOI=1) for 6 h and stained with anti-IFI16 antibody (red). DNA is visualized with DAPI (blue). Scale bars, 20 μm. (c–e) qRT-PCR analysis of IFI16 +/+ and IFI16 −/− HaCaT cells infected with HSV-1 (MOI=1) for 6 h. mRNA expression levels normalized to β-actin mRNA were determined for IFNb (c), ISG56 (d) and IL6 (e). (f) Secreted CCL5 protein from HaCaT cells infected with UV inactivated HSV-1 (MOI=5) for 24 h, quantified by ELISA. (g,h) qRT-PCR analysis of ISG56 (g) and CCL5 (h) mRNA expression in HaCaT cells infected with MVA (MOI=5) for 6 h. (i) ELISA quantitation of CCL5 protein in supernatants from HaCaT cells infected with MVA (MOI=5) for 24 h. (j) ELISA analysis of CCL5 protein from HaCaT cells infected with a Sendai virus (SeV) preparation containing defective viral particles (1:2,000 dilution) for 24 h. (k,l) qRT-PCR analysis of IFNβ (k) and ISG56 (l) mRNA expression in HaCaT cells infected with Sendai virus (SeV) at dilutions of 1: 20 000, 1: 2,000 and 1:200 for 6 h. (m) Primary human keratinocytes (NHEK) were transfected with a non-targeting (NT) or IFI16-targeting siRNA pool for 48 h. Protein expression was examined by Western blotting. (n,o) NHEK were treated with siRNA pools for 48 h, and infected with HSV-1 (MOI=1) for 6 h. IFN-β (n) and IL-6 (o) mRNA expression levels were quantified by qRT-PCR. (p,q) qRT-PCR analysis of IFN-β mRNA expression in MRC-5 human embryonic lung fibroblasts treated with siRNA pools for 48 h, and transfected for 6 h with 1 μg ml−1 HT DNA (p) or 100 ng ml−1 poly(I:C) (q). Data are representative of at least two independent experiments, and presented as mean values of biological triplicates, with error bars indicating s.d. *P<0.05, **P<0.01, ***P<0.001 Student's t-test.