Staurosporine sts

Plasmids encoding FLAG-tagged Keap1, HA-tagged Ub, and His-tagged Nrf2 were purchased from GeneChem (Shanghai, China). Plasmids were transfected into cells using Lipofectamine 2000 reagent (Life Technologies). The small interfering RNAs (siRNAs) (nontargeting control siRNA and siRNA targeting Keap1) used in this study were synthesized by RiboBio (Guangzhou, China). The siRNAs were transfected into cells using riboFECT CP (RiboBio) according to the manufacturer’s instructions. Ferrostatin-1 (Fer-1) (catalog number S7243), RSL3 (catalog number S8155), MG132 (catalog number S2619), chloroquine (NSC-187208) (catalog number S6999), and indomethacin (IND) (NSC-77541) (catalog numberS1723) were purchased from Selleck Chemicals. Reduced l-glutathione (GSH) (catalog number HY-D0187) and staurosporine (STS) (catalog number HY-15141) were purchased from MedChemExpress (MCE). Deferoxamine (DFO), N-acetylcysteine (NAC), and TNF-α were purchased from Sigma-Aldrich.

A normal hepatocyte line (L02) and HCC cell lines (Hep3B, HepG2, Huh7, and PLC) were purchased from Shanghai Institute of Cell Bank (Shanghai, China). Huh7 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, USA) supplemented with NaHCO₃ (1.5 g/L). HepG2 and PLC cells were grown in minimum essential medium (Gibco, Grand Island, USA) with NaHCO₃ (1.5 g/L) and sodium pyruvate. Cells were cultured with 10% fetal bovine serum (Pansera ES, Pan biotech GmbH, Germany), penicillin (U/ml), and streptomycin (0.1 mg/ml) at 37°C in a humidified atmosphere containing 5% CO₂. Cells were exposed to sorafenib (Solarbio, Beijing, China) for the indicated time and at the indicated concentration. Drug-resistant cell lines were established by stepwise selection of cells cultured in growth media with increasing concentrations of the drug over a period of 6 months. Erastin, staurosporine (STS), ferrostatin-1 (Fer-1), deferoxamine (DFO), ZVAD-FMK, and necrosulfonamide (NSA) were purchased from MedChemExpress (New Jersey, USA). sorafenib and pioglitazone (Pg) were purchased from Solarbio Biotechnology Company (Beijing, China).