Dual luciferase activities

The IFN-I production assay was performed as described previously (Xia et al., 2018 (link)). Briefly, HEK293T cells (1 × 10⁵ cells per well in a 24-well plate) were co-transfected with 10 ng of IFN-β promoter reporter plasmid, 4 ng of Renilla luciferase plasmid, 20-80 ng of viral protein expression plasmid using X-treme-GENE™ 9 transfection reagent with a ratio 1:2. Empty pXJ vector was used to ensure the same total amount (100 ng) of plasmids in each well. Cells were induced by co-transfection with 4 ng/well of stimulator expressing plasmids [(RIG-I (2CARD), MAVS, TBK1, IKKε, or IRF3)], same amount of empty pXJ vector was used as non-stimulated control. At 24 h post-transfection, the cells were assayed for dual-luciferase activities according to the manufacturer’s instructions (Promega). For the IFN-I signaling, HEK293T cells (1 × 10⁵ cells/well, 24-well plate) were co-transfected with 250 ng of ISRE promoter reporter plasmid, 20 ng of Renilla luciferase plasmid, and 230 ng of viral protein expression plasmid. At 16 h post-transfection, the transfected cells were treated with 1,000 units/ml of human IFN-α (Millipore). After another 8 h incubation, the cells were lysed and performed dual-luciferase reporter assays according to the manufacturer’s instructions (Promega). Luciferase signals were read by Cytation 5 (Bio Tek, Winooski, VT).