Transcriptional Activity of Nrf2 Using Dual-Luciferase Assay

Dual-luciferase report assay was conducted to assess the transcriptional activity of Nrf2. The human PINK1 ARE (5′-TGCTTGAGC-3′) and HMOX1 ARE (5′-CGGACCTTGACTCAGCAGAAAA-3′) were, respectively, inserted into the pGL3 vector (Promega, Madison, WI, USA) by Genepharma (Shanghai, China). The plasmid pRL-TK encoding Renilla luciferase was used as an internal control. Then, cells were co-transfected with pGL3 vector, pcDNA3.1-Nrf2, or internal control plasmid (pcDNA3.1-vector) by Lipofectamine 2000 reagent (Invitrogen, CA, USA) at 37 °C according to the manufacturer’s instruction in A549 cells. Additionally, the luciferase assay was performed 48 h after transfection using the Firefly/Renilla Dual-Luciferase Reporter Assay System (Promega, WI, USA) [18 (link),19 (link),20 (link)].