Regulation of NF-κB and TNF-α Promoter Activity

RAW264.7 cells (2 × 10⁵ cells/well) were seeded in 24-well plates. After 12 h, cells were co-transfected with 0.25 μg of Renilla-expressing plasmids (pRL-SV40-C; Beyotime Biotechnology, Shanghai, China) and either 1 μg of NF-κB binding site reporter plasmids (pNF-κB-TA-Luc; Beyotime Biotechnology, Shanghai, China) or 1 μg of TNF-α promoter reporter plasmids (pTNF-α-promoter-Luc; Beyotime Biotechnology, Shanghai, China) using TurboFect (Invitrogen, Carlsbad, USA). At 4 h post-transfection, cells were treated with PBS, 100 ng/mL ET(H351A), 100 ng/mL ET, 10 μM BAY 11-7082 (BAY), and 40 μM H89, in the presence or absence of 10 ng/mL LPS or 10 ng/mL TNF-α. Luciferase activity levels were determined using the Dual-Luciferase reporter assay system (Promega, Madison, WI, USA) using a microplate luminometer (GLOMAX96; Promega, Madison, WI, USA), following the manufacturer’s instructions. Firefly luciferase activity was normalized against Renilla luciferase activity.