Pirf3 luc

The luciferase assay for detecting IFN-β, NF-κB and IRF3 promoter activities were carried out as described before [21 (link)]. The plasmids pIFN-β-luc, pIRF3-luc, pNF-κB-luc, and pRL-TK were purchased from Promega. Briefly, the viral protein expression plasmids together with luciferase plasmids and pRL-TK were co-transfected into HEK293T cells. The pRL-TK vector provided constitutive expression of Renilla luciferase. After 24 h post-transfection, cells were mock- or treated with 200 ng Poly (I:C) (Sigma, China) for 12 h. The luciferase activity in cells lysates were detected using the Dual-Luciferase assay system (Promega, China) according to the manufacturer’s instructions.

Cells were cultured in a 6-well plate at a density of 2 × 10⁵ cells/mL for 24 h. pNF-κB-Luc (Stratagene, La Jolla, CA, USA), pIRF3-Luc (a kind gift from Dr. Katherine Fitzgerald, University of Massachusetts Medical School, Worcester, MA, USA), and pRL-TK control reporter vectors (Promega, Madison, WI, USA) were transfected using Lipofectamine 2000 according to the manufacturer’s protocol. After transfection, the cells were pretreated with various concentrations of 30 or 50 µM catalpalactone for 2 h and then stimulated with or without LPS (1 μg/mL) for 24 h. Luciferase activity was measured using the Dual-luciferase Reporter Assay System (Promega, Madison, WI, USA). The results are presented as the mean ± SD of three replicates for one representative experiment.