Luciferase reporter gene assay

DF-1 cells (~1 × 10⁵) were seeded in 24-well dishes and transfected with pGL3-LTR the following day using Lipofectamine 3000 (Life technologies). To normalize for transfection efficiency, the thymidine kinase (TK)-Renilla luciferase reporter plasmid (pRL-TK) was added to each transfection. At 6 h post-transfection, cells were treated with the inhibitors of the MAPK signaling pathways. Reporter luciferase activity was measured at 48 h post-transfection using the Dual-Luciferase Reporter Assay System (Promega) and a Tecan Infinite M1000 luminometer (Tecan, Maennedorf, Switzerland) according to the manufacturer’s instructions. Firefly luciferase activity was normalized against the activity of Renilla luciferase. Data are representative of three independent experiments, performed in triplicate.