Sulfasalazine

Human normal prostate stromal immortalized cell line WPMY-1 (BNCC100291) and PCa cell lines PC-3 (BNCC337715), Du 145 (BNCC338240), LNCaP (BNCC337703), and 22RV1 (BNCC100161) were bought from BeNa Culture Collection (Shanghai, China). Cells were prepared at 37℃ with 5% CO₂. Culture medium information was: PC-3, Du 145, WPMY-1 cell lines in DMEM-H (BNCC338068) plus 10% FBS; LNCaP, 22RV1 cell lines in RPMI-1640 plus 10% FBS (BNCC341471). Du 145 cells were passaged in DMEM (Sigma-Aldrich) plus 10% dialyzed FBS (Gemini Bio-Products) in the methionine dependence assay. Control medium was supplemented with 100 µM l-methionine (Sigma-Aldrich), 100 µM l-cysteine (Fisher Scientific), 1.5 µM vitamin B12, and 4 mM l-glutamine. Also, 200 µM dl-homocysteine (Sigma-Aldrich) was added to the Met-Hcy+ medium. For sulfasalazine treatment, sulfasalazine (HY-14655, medchemexpress, USA) was purchased and dissolved in DMSO, and was added to the culture medium at a final concentration of 200 µM for treating PCa cells. The same amount of DMSO was supplemented to the control group for treatment [33 (link)].

Aspc-1, HepG2, Panc02, and H22 cell lines were obtained from the KeyGEN Biotechnology Company (China). HT1080 and SW480 were obtained from the FuHeng BioLogy Company (China). HT1080 cancer cells were cultured in Eagle’s Minimum Essential Medium supplemented with 10% fetal bovine serum (FBS), glutamine (2 mM), penicillin (100 U/ml), and streptomycin (0.1 mg/ml). SW480, Aspc-1, HepG2, Panc02, and H22 were cultured in high Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, L-glutamine (4 mM), and penicillin (100 U/ml) and streptomycin (0.1 mg/ml). All cell lines were maintained in a humidified atmosphere containing 5% CO₂ at 37 °C and tested for mycoplasma prior to the commencement of experiments. Unless otherwise indicated, cell culture medium was changed every 3 days, and cells were passaged using 0.05% trypsin/EDTA. Erastin (#HY-15763), sorafenib (#HY-10201), sulfasalazine (#HY-14655), DON (#HY-108357), RSL3 (#HY-100218A), L-Buthionine-(S,R)-sulfoximine (BSO, #HY-106376A), ferrostatin-1 (#HY-100579), Z-VADFMK (#HY-16658), AICAR (#HY-13417), BafA1 (#HY-100558), N-Acetylcysteine (#HY-B0215), and Necrosulfonamide (#HY-100573) were purchased from MedChemExpress (USA). Compound C (#ab120843) was purchased from Abcam. Deferoxamine mesylate (#D9533) was purchased from Sigma-Aldrich.

Sulfasalazine (#HY‐14655) and ferrostatin‐1 (#HY100579) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Rapamycin (R0395) was purchased from Sigma‐Aldrich (St Louis, MO, USA). Antibodies targeting Beclin1 (3495S; Cell Signaling Technology, Danvers, MA, USA), LC3 (L7543; Sigma‐Aldrich) and GAPDH (sc‐47724; Santa Cruz Biotechnology, Santa Cruz, CA, USA) were also used.