Nrf2 Activation by 7-ML in HepG2 Cells

An antioxidant response element (ARE) promoter fragment (697 bp) of human the NQO1 promoter was extracted from the genomic DNA of HCT116 human colorectal cancer cells and inserted into the pGL3-Basic plasmid (Promega, Madison, WI, USA). HepG2 cells were seeded in a 24-well plate at a density of 6 × 10⁴, incubated for 24 h, and transiently cotransfected with the ARE-Luc reporter plasmid or the control plasmid pRL-SV40 using the TransFast™ reagent (Promega). Transfected cells were treated with 7-ML for 20 h and then with tBHP (tert-butyl hydroperoxide; 200 μM) for 2 h. Firefly and Renilla luciferase activities were then determined using a Dual-Luciferase reporter gene assay system (Promega). Nrf2 (nuclear factor-erythroid 2-related factor 2) luminescence signals were normalized versus SV40 (Renilla) luciferase activity. As a positive control for ARE-luciferase activity, sulforaphane was used [33 (link)].