Brusatol

Luciferase reporter assay was conducted on HepG2-ARE cells as described [18 (link), 20 (link)]. The cells were treated with samples for 12 h after serum starvation (0.5% FBS, 12 h). The luciferase activity, which corresponded to the ARE activity, was measured using a luciferase assay system (Promega) according to the manufacturer's instruction. Sulforaphane (Sigma-Aldrich), an isothiocyanate, was used as an ARE activator. Brusatol (Carbosynth Ltd., Newbury, Berkshire, UK), a quassinoid, was used as a specific inhibitor of the Nrf2 pathway [21 (link)]. The luminescence of the assay was detected and calibrated on total protein amounts. The data were then normalized against the control values.

EA.hy926 and HeLa cells (obtained from ATCC^® CRL-2922™, somatic cell hybrid established by fusing primary HUVEC with a thioguanine-resistant clone of A549; ATCC^® CCL-2, cervical cancer cells; passage No. 15-20; VA, USA) were cultured in Dulbecco’s Modified Eagle Medium; 4.5 g/L Glucose (DMEM; Thermo Fisher Scientific, MA, USA) containing 10% FCS (0.1% FCS under starvation) and 100 U/mL Penicillin and 100 µg/mL Streptomycin in a 37 °C humidified incubator in the presence of 10% CO₂. EA.hy926 reporter cells (NRF2-ARE-mCherry fluorescence based, see Figure 4a) were generated by transfection of plasmid (CS-13227-LvGN01; GeneCopoeia, MD, USA) by FuGene HD Transfection reagent (Promega, WI, USA). Transfection efficiency was improved by transfection of linearized and shortened plasmid (NsbI-AjuI-digest; NEB, MA, USA). Positive cells were selected by puromycin treatment (0.25 µg/mL) and sorted by flow cytometry (BD FACS Aria2; NJ, USA; more than 4-fold over background) after stimulation with 20 ng/mL Phorbol 12-myristate 13-acetate (Sigma-Aldrich, MO, USA). On 6-well dishes, 3 × 10⁵ cells per well were seeded and, where indicated, cells were incubated with different NRF2-inducers (5 µM, 10 µM sulforaphane; LKT Laboratories, MN, USA; and 4 µM, 8 µM falcarinol; Cayman chemical, MI, USA) or inhibitors (10 nM, 30 nM, 100 nM, 300 nM brusatol; Carbosynth, UK).

A human hepatoma cell line HepG2 was obtained from the Korean Cell Line Bank (KCLB; Seoul, Korea) and propagated in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (all from Invitrogen/Life Technologies). The cells were then transfected with pGL4.37[luc2P/ARE/Hygro] vector (Promega, Madison, WI, USA) using TurboFect transcription reagents (Thermo Fisher Scientific, Waltham, MA, USA). The transfectant carrying an ARE-luciferase construct was named HepG2-ARE and maintained in its growth medium containing 0.4 mM hygromycin (Sigma-Aldrich, St. Louis, MO, USA).
To measure the transcriptional activity of Nrf2, luciferase reporter assay was conducted on HepG2-ARE cells as described by Kim et al. [26 (link)]. The cells were treated with samples for 12 h after serum starvation (0.5% FBS, 12 h). The luciferase activity, which corresponded to the ARE activity, was measured using a luciferase assay system (Promega) according to the manufacturer’s instruction. Sulforaphane (Sigma-Aldrich), an isothiocyanate, was used as an ARE activator [27 (link),28 (link)]. Brusatol (Carbosynth Ltd., Newbury, Berks, UK), a quassinoid, was used as a specific inhibitor of the Nrf2 pathway [23 (link)]. The luminescence was detected using a TD-20/20 luminometer (Turner Designs, Sunnyvale, CA, USA), and calibrated to total protein amounts. The data were then normalized against the control values.