4.9. Luciferase Reporter Assay

Reporter cells were seeded (1 × 104 cells/well) in 96-well plate, then treated with indicated concentrations of compounds for 18 h. At the assay time point, resazurin (Cayman Chemical, Ann Arbor, MI, USA) was added to a final concentration of 0.1 mg/mL and further incubated for 4 h at 37 °C. Fluorescence of the reduced resazurin (ex/em: 530/590 nm) was measured from the culture supernatant by using a Synergy HT Multi-Mode Reader (BioTek, Winooski, VT, USA) to determine cell viability. The cells were then harvested for luciferase activity measurements according to the manufacturer’s protocol (Promega Corporation, Madison, WI, USA). Relative luciferase activity was calculated by normalizing luciferase activity to cell viability. For GNMT promoter activity assay treatment, data were presented as the fold to DMSO solvent control; for NRF2-activity assay, DMSO solvent control was used as 100% activity.