Ef 24

HONE-1 cells were obtained from the Food Industry Research and Development Institute (Hsinchu, Taiwan). NPC-39 and NPC-BM, derived from patients with NPC [38 (link)], were kind gifts from Dr. MK Chen, Department of Otolaryngology, Changhua Christian Hospital, Changhua, Taiwan. The cell culture was maintained in RPMI-1640 medium at 37 °C in a humidified atmosphere of 5% CO₂. EF-24 was purchased from Sigma-Aldrich (St. Louis, MO, USA) and prepared in dimethyl sulphoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA). Where indicated, the cells were pretreated with EF-24 for 1 h and then incubated with 50 ng/mL TPA (Sigma-Aldrich, St. Louis, MO, USA) for 23 h, followed by the subsequent experiments.

EF24, N-acetylcysteine (NAC), catalase and rhTrxR1 protein were purchased from Sigma (St. Louis, MO). Erastin was purchased from Selleck Chemicals. Human gastric cancer cell lines SGC-7901, BGC-823 and KATO III, normal Human Gastric Epithelial Cell Line (GES-1) and normal Rat Kidney Proximal Cell Line (NRK-52E) were purchased from the Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences. The cells were routinely cultured in RPMI 1640 medium (Gibco, Eggenstein, Germany) containing 10% heat-inactivated fetal bovine serum (Gibco, Eggenstein, Germany), 100 units/mL penicillin, and 100 μg/mL streptomycin in a humidified cell incubator with an atmosphere of 5% CO₂ at 37°C. Antibodies including anti-Bcl-2, anti-Bax, anti-cleaved PARP, anti-TrxR1, anti-GAPDH, goat anti-mouse IgG-HRP and donkey anti-rabbit IgG-HRP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies including anti-CHOP, anti-ATF4, anti-p-eIF2α and anti-eIF2α were purchased from Cell Signaling Technology (Danvers, MA). FITC Annexin V apoptosis Detection Kit I and Propidium Iodide (PI) were purchased from BD Pharmingen (Franklin Lakes, NJ).

EF-24 (3,5-bis[(2-fluorophenyl)methylene]-4-piperidinone) and DL-sulforaphane were obtained from Sigma-Aldrich (Saint Louis, MO, USA) and dissolved in DMSO. The residual concentration of DMSO in growth medium was approximately 0.1–0.2%. Catalase (CAT) from bovine liver was dissolved in 25 mM Tris/HCl buffer, pH = 7.0. Cell treatment was done using 50 units of CAT per mL. N-acetylcystein (NAC) was dissolved in distilled water. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Zosuquidar trihydrochloride (ZSQ; LY335979) and Ko143 (3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-(2-methylpropyl)-1,4-dioxopyrazino-[1′,2′:1,6] pyrido [3,4-b]indole-3-propanoic acid 1,1-dimethylethyl ester were obtained from Enzo Life Sciences AG (Lausen, Switzerland).