2.1. Transactivation assay

For AhR-mediated transactivation, HepG2 cells were plated at 7.5 × 10⁴ cells/well in a 24-well plate. Cells were transiently transfected with 200 ng of the reporter vector pLightAhRE, 100 ng of pCMVXL4AhR, and 100 ng of pGL4.70 (Promega, Madison, WI, USA), a vector encoding the human RENILLA luciferase gene. At 24 h post-transfection, cells were stimulated 18 h with Pelargonidin 10 and 50 μM (Merk Group, Darmstadt, GE). Dose-response curves were performed in HepG2 cells transfected with AHR, as described above, and then treated with increasing concentrations of Pelargonidin (1, 2.5 5, 10, 12.5, 25, 50, 75, 100 μM).

At 18 h post-stimulation, cellular lysates were assayed for luciferase and RENILLA activities using the Dual-Luciferase Reporter assay system (Promega, Madison, WI, USA). Luminescence was measured using Glomax 20/20 luminometer (Promega, Madison, WI, USA). LUCIFERASE activities (RLU) were normalized with RENILLA activities (RRU).