Ml210

The human prostate cancer cell lines LNCaP, C4‐2B, VCaP, PC3 were purchased from the American Type Culture Collection and cultured following ATCC's instructions. The LNCaP‐AI cells were established by culturing the LNCaP cells in media with 10% charcoal‐stripped fetal bovine serum (CSS; Hyclone). Ceapin‐7 and A‐23187was purchased from Sigma. All cells were maintained at 37°C in a humidified incubator with 5% CO₂. RSL3, ML210, ferrostatin‐1, liproxstatin‐1, zVAD‐fmk, necrostatin‐1, Erastin, PAF, and Enzalutamide were purchased from Selleck Chemicals.

Erastin, RSL3, ML210, and liproxstatin-1 (LIP-1) were purchased from Selleckchem. Cyclophosphamide, paclitaxel, U18666A, cisplatin and 5-fluorouracil were obtained from TargetMol. Rosiglitazone, MβCD, avasimibe, cholesterol, betulin and IFN-γ were obtained from MedChemExpress. LDL, ox-LDL, HDL and dil-LDL were obtained from Guangzhou Yiyuan Biological Technology Co., Ltd. Soy phospholipid mixture, methyl linoleate, linoleic acid, lovastatin and adriamycin were purchased from Sigma.

Cells (1 × 10⁴) were seeded in a 96-well plate. The next day, the cells were treated with the indicated agents RSL3 (#19288, Cayman Chemicals), erastin (#17754s, Cayman Chemicals), ML210 (#S0788; SelleckChem), ML162 (#S4452; SelleckChem), and liproxstatin-1 (#7699, SelleckChem) for 24 hours, and cell viability was assessed with an MTT [3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide] colorimetric assay. For the combination of liproxstatin-1 and ferroptosis activators, liproxstatin-1 was added 30 min before the activators. For the combination of brusatol and RSL3 experiments, brusatol was added 3 hours before adding RSL3. For reverse transcription qPCR (RT-qPCR) experiments, cells seeded 24 hours before treatment with brusatol for 3 hours or luteolin (S2320, SelleckChem) for 24 hours with varying concentrations as indicated in figure legends.