Nrf2 reporter assay

An Nrf2 reporter assay based on the dual luciferase system was performed as previously described [23,29]. The plasmid pNQO1-ARE (antioxidant response element)-Fluc, provided by Yamamoto M., expressed a Firefly luciferase gene driven by Nrf2 activation, while the plasmid pRL-TK-Rluc vector (Promega, CA, USA) expressed a Renilla luciferase gene driven by the herpes simplex viral thymidine kinase promoter as an internal control. MDCK cells were seeded in a 24-well plate at 1 × 10⁵ cells/well and transfected with pNQO1-ARE-luc (0.25 μg) and pRL-TK-Rluc (0.25 μg). At 24 h post-transfection, the cells were treated with 12.5 μM cyclobakuchiols A–D in the infection medium at 37°C under 5% CO₂. DMSO (0.125%) and 12.5 μM 1 were used as negative and positive controls, respectively. Total RNA was extracted from the MDCK cell lysates after 24 h of incubation. The levels of Firefly and Renilla luciferase mRNAs were analyzed by RT-qPCR and normalized to that of β-actin mRNA.