Transfection and luciferase reporter assay

HeLa cells (2 × 10⁴ cells per well) were seeded into 96-well plates and were transfected after 24 h with expression vectors and luciferase reporter genes by using Lipofectamine 3000 (Invitrogen, USA). The transfection procedure was performed as described in a previous study⁷⁴. In brief, 20 ng/well pGL3/NF-κB-luc reporter gene combined with 25 ng/well pcDNA3.1/FLAG-MyD88, or 10 ng/well pcDNA3.1/FLAG-TRAF6, or 10 ng/well pcDNA3.1/FLAG-p65 plasmids, and PC2AOE-3×FLAG-NOC4L expression plasmids in different amounts (0, 30, 60 ng) or PC2AOE-GFP plasmid, was co-transfected into the cells. Similarly, 20 ng/well pGL3/NF-κB-luc reporter genes combined with 20 ng/well pcDNA4.0-TRIF and PC2AOE-3×FLAG-NOC4L expression plasmids in different amounts (0, 25, 50, and 100 ng) or PC2AOE-GFP plasmid were co-transfected into the cells. In addition, 10 ng/well of the phRL-TK reporter plasmid (Promega, USA) was co-transfected to allow normalization of data for transfection efficiency. The total amount of DNA per transfection was kept constant at 150 ng by adding the empty vector PC2AOE-3×FLAG. After transfection for 24 h, luciferase activity was measured using a Dual-Luciferase reporter assay system (Promega, USA) following the instructions provided by the manufacturer. The luciferase activity was normalized against Renilla luciferase activity. All reporter assays were completed in 4 repeat wells, and data were presented as mean ± SEM.