Pifn β 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.

After overnight culture in 24‐well plate, A549 cells were co‐transfected with 1,000 ng pIFNβ‐Luc (Siu et al, 2009) and 1 ng TK‐Nano‐Luc (Promega) per well. At 24 h post‐transfection, cells were further transfected with Poly I:C (0 or 50 μg/ml, InvivoGen) and were simultaneously treated with the indicated concentrations of AOAA (Sigma‐Aldrich, CAS No. 2921‐14‐4) and incubated at 37°C for 16 h. Subsequently, the cells were lysed for luciferase assay using Dual‐Glo Luciferase Assay System (Promega) in a Victor X3 Multilabel reader (PerkinElmer).
A549‐Dual™ Cells and A549‐Dual™ KO‐RIG‐I Cells (InvivoGen) were transfected with Poly I:C and treated with the indicated concentrations of AOAA for 16 h. Cell‐free media were used for luciferase assay using QUANTI‐Luc™ (InvivoGen). A549 cells were transfected with GLDC siRNA or scrambled siRNA (50 nM, Thermo Fisher Scientific) using Lipofectamine RNAiMAX (Thermo Fisher Scientific). Two days after siRNA transfection, the cells were further transfected with Poly I:C of 50 μg/ml and maintained in DMEM for 24 h. The cells and cell‐free media were then harvested for detection of mRNA expression of GLDC and IFNα by RT–qPCR assay and measurement of IFNα production by VeriKineTM Human IFN Alpha ELISA Kit (PBL Assay Science), respectively.