Fer 1

RSL3 (#HY-100218A) and Fer-1 (#HY-100579) were purchased from MedChemExpress (MCE, Monmouth Junction, NJ, USA). Sodium butyrate (NaB, #S1999) was purchased from Selleck Chemicals, Houston, TX, USA. Dimethyl sulfoxide (DMSO, Solarbio, Beijing, China, #D8371) was used to prepare the stock solution of drugs. FFAs was purchased from Shanghai Keshun Science and Technology co., Ltd. (Shanghai, China). Fetal Bovine Serum (FBS, # FSD500) was purchased from Excell Bio. DCFH-DA (#S0033S) and DAPI (#C1002) were purchased from Beyotime Bio (Shanghai, China). Alanine aminotransferase (ALT, #C009-2-1), aspartate aminotransferase (AST, #C010-2-1), total cholesterol (TC, #A111-1-1), triglyceride (TG, #A110-1-1), malondialdehyde (MDA, #A003-4-1), glutathione peroxidase (GSH-pX, #A005-1-2), and glutathione (GSH, #A006-2-1) were purchased from Nanjing Jiancheng Bio (Nanjing, China). Transferrin receptor (TFRC, # bs-41331R), ferritin heavy chain 1 (FTH1, #bs-5907R), nuclear receptor coactivator 4 (NCOA4, #bs-19051R), cyclooxygenase-2 (COX-2, #bs-10411R), GPX4 (#bs-3884R), and GAPDH (#bs-41373R) were purchased from Bioss Bio (Beijing, China). Solute carrier family 7 member 11 (SLC7A11 or xCT, #26864-1-AP) was purchased from Proteintech Bio (Rosemont, IL, USA).

Fer‐1 was obtained from MedChemExpress. Dexamethasone (Dex) was purchased from Meilunbio. Soybean lecithin, cholesterol, and 1,2‐distearoyl‐sn‐glycero‐3‐phosphoethanolamine‐N‐[methoxy (polyethylene glycol)‐2000] (DSPE‐mPEG) were obtained from AVT Pharmaceutical Technology. Green fluorescent protein‐transduced human umbilical vein endothelial cells (HUVEC‐GFP) were obtained from Beyotime. Human corneal epithelial cells (HCECs) were obtained from the ATCC. Calcein‐AM/PI double stain kit was purchased from Yeasen. A Cell Counting Kit‐8 (CCK‐8) was purchased from Dojindo. Recombinant human vascular endothelial growth factor (rhVEGF) was purchased from Peprotech. Matrigel Matrix Growth Factor Reduced was obtained from BD Bioscience. Rabbit anti‐4 hydroxynonenal (4‐HNE), anti‐glutathione peroxidase 4 (GPX4), anti‐acyl‐CoA synthetase long‐chain family member 4 (ACSL4), anti‐CD31, and anti‐α‐smooth muscle actin (α‐SMA) antibodies were purchased from Abcam. Rabbit anti‐IL‐1β and anti‐IL‐6 antibodies were purchased from Affinity Bioscience. PrimeScriptTM RT Master Mix was acquired from Takara. ChamQ SYBR qPCR Master Mix was acquired from Vazyme.

Renal cell cancer cell lines 786-O and A498 were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, GIBCO, MA, USA) under 5% CO₂ at 37°C. For glucose starvation treatment, cells were cultured in normal medium for 24 h and washed twice with PBS, and the medium was replaced with a glucose-free medium supplemented with 10% FBS.
Cells were treated with the ferroptosis inducer erastin (1.5 μM) (MedChemExpress, China) for 24 h, the ferroptosis inhibitor ferrostatin-1 (2 μM) (Fer-1, MedChemExpress) for 24 h, the AMPK inhibitor Compound C (COM C) (10 μM) (MedChemExpress) for 24 h, the AMPK activator AICAR (2 mM) (MedChemExpress) for 24 h, Pifithrin-α (5 μM) (PFT-α, MedChemExpress) for 24 h, the apoptosis inhibitor Z-VAD-FMK (20 μM) (MedChemExpress), and the necroptosis inhibitor necrostatin-1 (2 μM) (Nec-1, MedChemExpress).

To establish in vitro models of NASH, AML12 and HepG2 cells were exposed to free fatty acids (FFAs), including oleic acid and palmitic acid (OA: PA = 2:1), for 24 h at a concentration of 300 µM. Simultaneously, cells were separately treated with erastin (8 µM) and Fer-1 (4 µM), which were obtained from Med Chem Express (Shanghai, China). We also established an HO-1 knockdown model by transfecting small interfering RNA (siRNA) into cells while inducing HO-1 overexpression with pcDNA3.1 HO-1 plasmid (Gene Pharma, Shanghai, China).
Cells were divided into 6 groups to investigate the role of HO-1 in hepatocytes. Namely, the control group (NC group, nontreatment), model group (FFA group, 300 µM FFA treatment), ferroptosis inducer group (erastin group, 8 µM erastin intervention), FFA combined with ferroptosis inhibitor group (Fer-1 group, 4 µM Fer-1 intervention, 300 µM FFA treatment), HO-1 knockdown group (kdHO-1 group, HO-1 siRNA transfection, 300 µM FFA treatment), HO-1 overexpression group (oeHO-1 group, pcDNA3.1 HO-1 plasmid transfection, 300 µM FFA treatment).

Briefly, 1 × 10⁴ pancreatic cancer cells in 100 μL culture media were seeded in a 96-well plate, then added with ferroptosis inhibitor Ferrostatin-1 (Fer-1, 10 μmol/L; MedChemExpress, Shanghai, China), and cultured at 32°C. Then, the cells were incubated with CCK-8 solution (10 μL/well, Beyotime) for 4 h at 37°C. The optical density in each well was quantified at 450 nm wavelength with a microplate reader.