(A) ISD DNA of different lengths was transfected into MEFs and the transcription of Cxcl10 was assayed by qPCR 6 hr later. (B) Double stranded oligonucleotides (bio-ISD), concatenated ISD DNA (bio-concatamers), genomic vaccinia virus DNA (bio-VACV), genomic E. coli DNA (bio-E. coli), poly (dA:dT) or the RNA analogue poly (I:C) were biotinylated and transfected into HEK293 cells. Following affinity purification of proteins from cytoplasmic extracts using streptavidin agarose, the bound proteins were analysed by SDS-PAGE and immunoblotting. AP; affinity purification. (C) Primary MEFs of the indicated genotype were transfected with 10 μg/ml of the same (non-biotinylated) nucleic acids as in (A) followed by qRT-PCR analysis measuring induction of Cxcl10 mRNA 6 hr later. (D) Wild type and Prkdc−/− transformed MEFs were transfected with DNA (10 μg/ml, left panel) or stimulated with LPS (100 ng/ml, right panel) and the level of transcription of cxcl10 was measured at the indicated times post stimulation. (E) Levels of Cxcl10 and Ifnβ were measured by ELISA from the supernatants of primary wild type and Prkdc−/− MEFs at passage 1, 24 hr after transfection with DNA or poly (I:C). (F),(G) Primary wild type and Prkdc−/− MEFs at passage 1 were transfected with DNA or poly (I:C) and the level of induction of (F) Ifnb and Il-6 and (G) ccl4 and ccl5 mRNA was measured by qRT-PCR 6 hr later. (H) MEFs expressing Ku80 or lacking Ku80 were transfected with DNA and the transcription of Cxcl10 or Il6 was measured by qPCR 6 hr later. (I) Primary murine skin fibroblasts (MSF) from wild type adult mice or those lacking both Ku genes were transfected with DNA or poly (I:C) and the level of Ifnb induction was measured 6 hr later by qRT-PCR. (J) Xrcc5+/+/Trp53−/− and Xrcc5−/−/Trp53−/− MEFs were transfected with an expression plasmid encoding Ku80 or an empty vector (EV) control and Cxcl10 production was measured 24 hr later by ELISA. *** p<0.001, ** p<0.01, * p<0.05, n ≥ 3, error bars ± SEM, ns; non-stimulated.