N acetylcysteine

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.

Primary antibodies including p62 (ab109012), LC3 (ab62721), B-cell lymphoma 2 (Bcl-2, ab196495), p16 (ab51243), p21(ab109199) and β-actin (ab6276) were acquired from Abcam (Cambridge, MA, USA). Primary antibodies against p-AMPK (50081), AMPK (5831), Bax (2772), and p53 (2524) were purchased from Cell Signaling Technologies (CST; Danvers, MA, USA). Primary antibodies against cleaved caspase-3 (C-caspase-3, WL01992) were purchased from WanleiBio (Shen Yang, China). Goat anti-rabbit IgG (7074) and goat anti-mouse IgG (7076) secondary antibodies were purchased from CST (Danvers, MA, USA). Cell culture reagents were purchased from Gibco (Grand Island, NY, USA). D-(+)-Glucose was purchased from Sigma-Aldrich (St. Louis, MO, USA). AICAR and Acetylcysteine also called N-acetyl cysteine (NAC) were acquired from MedChemExpress (MCE; Manmouth Junction, NJ, USA).

The following chemical reagents were used in this study: Oxaliplatin (#HY‐17371; MedChemExpress, NJ, USA), 5‐Aza‐dC (#A3656; Sigma–Aldrich, MO, USA), Trichostatin A (#V900931; Sigma–Aldrich), chloroquine (#HY‐17589A; MedChemExpress), cycloheximide (#S7418; Selleck Chemicals, TX, USA), MG132 (#M7449; Sigma–Aldrich), RO‐3306 (#S7747; Selleck Chemicals), erastin (#S7242; Selleck Chemicals), RSL3 (#S8155; Selleck Chemicals), ferrostatin‐1 (#S7243; Selleck Chemicals), deferoxamine (#HY‐B1625; MedChemExpress), N‐acetyl‐cysteine (#HY‐B0215; MedChemExpress), Z‐VAD‐FMK (#V116; Sigma–Aldrich), necrostatin‐1 (#S8037; Selleck Chemicals), Rosiglitazone (#HY‐17386; MedChemExpress), and liproxstatin‐1 (#HY‐12726; MedChemExpress).

Cisplatin, 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), Sulforhodamine B (SRB) and crystal violet were purchased from Sigma Aldrich (St. Louis, MO) and #43 was obtained from the NCI Developmental Therapeutics Program. MK-2206 was purchased from BioVision (Mountain View, CA). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), JC-1 dye, 2-NBDG and propidium iodide (PI) were obtained from Invitrogen Life Technologies (Carlsbad, CA). U0126 was purchased from Cell Signaling Technologies (Boston, MA). N-acetylcysteine (NAC) was purchased from MedChemExpress (Monmouth Junction, NJ). Stock solutions of #43, DCFH-DA, MK-2206 and U0126 were prepared in DMSO, Cisplatin and MTT were dissolved in phosphate-buffered saline (PBS), SRB solution was prepared in 1% acetic acid, 2-NBDG, PI and NAC were dissolved in water, and crystal violet was dissolved in 20% methanol.

Purified erianin (>98%) (Cat: B20844) was gained from Shanghai Yuanye Biological Co., Ltd. All antibody follow-up experiments used were anti-xCT (Cat: ab175186), anti-Glutaminase (Cat: ab93424), anti-GPX4 (Cat: ab125066), anti-Heme Oxygenase-1 (Cat: ab189491) were from Abcam (Cambridge, United Kingdom), anti-FTH1 (Cat: 4393S), anti-NRF2 (Cat:12721S), anti-GAPDH (Cat: 5174S) were provided by Cell Signaling Technology (Danvers, United States). And inhibitors mentioned in experiments were deferoxamine (Cat: S5742) (Selleck, Houston, United States), chloroquine (Cat: C6628) (Sigma-Aldrich, St. Louis, United States), Z-VAD-FMK (Cat: HY-16658B), N-Acetylcysteine (Cat: HY-B0215), necrostatin-1 (Cat: HY-15760), L-Glutathione reduced (Cat: HY-D0187), TBHQ (Cat:HY-100489) were obtained from MedChem Express (New Jersey, United States), pLVX-U6-NRF2-shRNA1-PGK-EGFP-E2A-Puro (Cat:p24452) (miaolingbio, Wuhan, China).

Mito-LND, N-Acetylcysteine (NAC) and ERK1/2 activator C16-PAF were purchased from MedChemExpress (Shanghai, China). Mito-LND and C16-PAF were dissolved in DMSO to generate stock solution (10 mM), and N-Acetylcysteine was dissolved in DMSO to produce stock solution (500 mM). These inhibitors were diluted to different concentrations in DMEM medium before use.
Primary antibodies against c-p-Raf (#9421), p-MEK1/2 (#9154), p-p90RSK (#9346), ERK1/2 (#4695), p-ERK1/2 (#4376), CDC2 (#28,439), CDK4 (#12,790), CDK6(#3136), Bax (#2772), Bcl-2 (#4223), cyclin D1 (#2922), cyclin B1 (#4138), GAPDH (#97,166) were purchased from Cell Signaling Technology (CST, MA, USA).

TEOA was obtained from college of pharmacy, Zhejiang University (Zhejiang, China) and dissolved in DMSO. N-acetylcysteine (NAC), Chloroquine (CQ), 3-Methyladenine (3MA) and Rapamycin were purchased from Medchem Express (MCE, United States). DAPI was purchased from Beyotime (Shanghai, China). The following primary antibodies were used: anti-GAPDH (Abcam ab181602) (1:2,000), anti-Catalase (Abcam ab76024) (1:1,000), anti-MnSOD (Abcam ab68155) (1:2,000), anti-LC3 (Sigma L7543) (1:1,000), anti-p-mTOR (Abcam ab109268) (1:2,000), anti-mTOR (Abcam ab134903) (1:1,000), anti-p-p70S6K (Abcam ab131436) (1:1,000), anti-p70S6K (Abcam ab32529) (1:1,000), anti-p-S6 (CST 4858S) (1:2,000), anti-S6 (CST 2217s) (1:1,000), anti-p-4EBP1 (CST 9459S) (1:1,000), anti-4EBP 1 (CST 9644S) (1:1,000), anti-ATG5 (Abcam ab68155) (1:2,000). The related HRP-conjugated secondary antibody was purchased from Beyotime (Shanghai, China).