Nrf2-luciferase reporter MCF7 stable cells (Signosis, Inc.) were grown in high glucose DMEM (Hyclone, Logan, UT, USA) supplemented with 10% FBS, 1% antibiotic-antimycotic solution, and 75 µg/mL geneticin. A 100-µL cell suspension was plated with a seeding density of 20,000 cells/well. The plate was incubated at 37 °C with 5% CO2 for 24 h prior to treatment. Cells were treated with compounds at half-log dilution, ranging from 0.032–100 µM (0.5% DMSO). DMSO (0.5%) and tBHQ (0.01–32 μM) served as solvent and positive controls, respectively. After 8-, 12-, and 16-h incubation, the medium was removed, and cells were washed with 100 µL 1X PBS. Lysis buffer (Signosis, Inc.) was added, and the plate was further incubated for 15 min at room temperature. A 100-µL firefly luciferase substrate (Signosis, Inc.) was added, and luminescence was measured. Nrf2 fold activation of treated cells was assessed relative to the solvent control. The assay was done in two independent experiments in triplicate.
For the cytotoxicity counter-screen, Nrf2-luciferase reporter MCF7 stable cells were grown, seeded, and treated identically with the reporter assay. Cell viability was done using the tetrazolium reagent, as indicated previously.
For the cytotoxicity counter-screen, Nrf2-luciferase reporter MCF7 stable cells were grown, seeded, and treated identically with the reporter assay. Cell viability was done using the tetrazolium reagent, as indicated previously.