4.10. NFκB Gene Reporter Luciferase Assay

pAECs grown at 80% confluence in six-well plates were co-transfected with 500 ng of pNF-κB-Firefly Luciferase plasmid (Agilent) expressing luciferase under control of NF-κB responsive elements and 50 ng of pRL-TK-Renilla-Luciferase expression vector (Promega, Charbonnières-les-Bains, France), which allowed the normalization of the transfection efficiency. Transfections were performed using 3.75 µL of Lipofectamine 3000 (Fisher Scientific, Illkirch-Graffenstaden, France) and 2 μL of reagent P3000 (Fisher Scientific, Illkirch-Graffenstaden, France) per well. Cells were treated with DMSO or CSC (100 µg/mL), whether combined or not with RAP (12.7 µg/mL), 24 h after the transfection for 48 h. Firefly and Renilla luciferase activities were then measured with a FB12 luminometer (Berthold, Thoiry, France) in the cellular extracts using the dual-luciferase reporter assay system (Promega, Charbonnières-les-Bains, France), according to the manufacturer’s instructions. This experiment was repeated three times (each condition in duplicate).