Antioxidant response dual-luciferase reporter assay

A Cignal Antioxidant Response dual-luciferase assay (Qiagen) was used to assess the activation of the Nrf2/Nrf1 signal-transduction pathway in usnic acid-treated HepG2 cells. The assay was performed according to the manufacturer’s protocol (G. transfection and treatment protocol for reporter assay + peptide/recombinant protein). Briefly, HepG2 cells at a density of 3 × 10⁴ cells per well were reverse transfected with Cignal reporter, Cignal negative control, or Cignal positive control plasmids using Lipofectamine^® 2000 Transfection Reagent (Thermo Fisher Scientific, Waltham, MA). After transfection and recovery for 24 h, cells in 6 replicate wells were treated with various concentrations of usnic acid for an additional 6 h. Firefly and Renilla luciferase activities were determined using a Dual-Luciferase^® Reporter Assay System (Promega Corporation, Madison, WI).