Nrf2 Activation Assay in HCT116 Cells

We acknowledge the gift of the pTRAF plasmid from Elias Arnér, Division of Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden. Colorectal carcinoma cell line HCT116 transformed with a pTRAF plasmid [18 (link),33 (link)] were cultured in Dulbecco’s modified Eagle’s (DMEM) supplemented with 10% fetal bovine serum at 37°C in 5% CO₂, 1% of penicillin/streptomycin and 0.1% of amphotericin B (Biochrom, United Kingdom). Cells were seeded in 96-well culture plates at a 1.0 x10⁴ cells/cm², and adhesion allowed for 24 hours. Portoamides were tested on a concentration-response assay (up to 10 μM) and incubated on an Incucyte® ZOOM Live-Cell Analysis System (Essen Instruments, Ann Arbor, MI). For the duration of 48h, Nrf2 activity was read at λex = 585 nm, emission filter: 625–705 nm, and auranofin (6 μM) was used as a positive control to confirm Nrf2 activity [18 (link)].