Fig. 4.

Induction of NIK via alternative NF-κB pathway-activating ligands or pharmacological reagents enhances the DNA pathway. a, b ELISA for IFN-β in supernatants collected from WT MEFs treated with DMSO (NT), LT-βR agonistic antibody (2 μg/mL) (a), or with SMAC mimetic (SM) (1 μg/mL) (b) and subsequently transfected with B-DNA (500 ng/mL) for 24 h. Data are means ± SEM of one experiment run in duplicates out of two independent experiments. c–e Q-PCR of IFN-β mRNA levels in BMDMs (c), A549 cells (d), or PBMCs (e) pretreated with DMSO (NT) or SM (1 μg/mL) for 16 h followed by B-DNA transfection (100 ng/mL) for 4 h. Data are means ± SEM of one experiment run in triplicates out of 2–3 independent experiments. f HSV-1 GFP virus populations at the indicated time points in WT MEF cells treated with SM (1 μg/mL), LT-βR agonistic Ab (2 μg/mL), or a DMSO control (NT) for 12 h and subsequently infected with HSV-1 GFP (MOI 0.1). Data are means ± SEM of one experiment run in duplicates out of two independent experiments. g Immunoblot analysis of STING phosphorylation (Ser 366) in THP-1 monocytes pretreated with vehicle control (DMSO) or SM (1 μg/mL) for 16 h followed by B-DNA transfection (1 μg/mL) for the indicated time points. Results are representative of two independent experiments. h Immunoblot analysis of IRF3-STING interactions in WT MEFs pretreated with DMSO or SM (1 μg/mL) for 16 h and subsequently transfected with B-DNA (2 μg/mL) for the indicated time points. Results are representative of two independent experiments. i Immunoblot analysis of IRF3 and TBK1 activation in A549 cells pretreated with vehicle control (DMSO) or SM (1 μg/mL) for 16 h followed by B-DNA transfection (2 μg/mL) for 4 h. Results are representative of three independent experiments. p < 0.05 is considered significant