Erastin

Cadmium chloride (CdCl₂, 202908), NAC (30498), and DFO (D9533) were obtained from Sigma-Aldrich (Sigma, St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM, Gibco, 12800-017), fetal bovine serum (FBS, Gibco, 10437-028), Trizol (Ambion, 15596026), and trypsin-EDTA (25200072) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). The ROS detection kit (S0033S), Mito-Tracker Red fluorescent probe (C1035), and Bicinchoninic acid (BCA) protein assay kit (P0012) were from Beyotime Biotechnology Co., Ltd. (Beyotime, Shanghai, China). The MDA (A003-1-2) and GSH detection kits (A006-2-1) were purchased from Nanjing Jiancheng Bio-Engineering Institute Co., Ltd. (Nanjing, China). The Cell Counting Kit-8 (CCK-8, A311-01) was purchased from Vazyme Biotechnology Co., Ltd. (Vazyme, Nanjing, China). The PrimeScrip^TM RT Reagent Kit (RR037A) and SYBR Green™ Premix Ex Taq™ (RR390A) were obtained from Takara Biomedical Technology Co., Ltd. (Takara, Beijing, China). The RIPA lysate (20101ES60) was obtained from New Cell & Molecule Biotechnology Co., Ltd. (NCM, Suzhou, China). The Fer-1 (HY-100579) and Erastin (HY-15763) were purchased from MedChemExpress LLC (MCE, Princeton, NJ, USA). All chemicals were of the highest purity grade available.

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).

Macrophages were implanted in a 96-well plate and cultured for 24 h at 37 °C. After macrophages had undergone different treatments at specific times, the supernatant was removed, and then the cells were washed with PBS three times. 100 μL 1640 medium containing 10 μL Cell Counting Kit-8 (CCK-8, Byotime, Beijing, China) was added into each well. The cells were incubated for 1 h at 37 °C until they turned orange and then measured at an absorbance of 450 nm. All drugs and corresponding treatment concentration: erastin (HY-15763), 10 μM; RSL3 (HY-100218A), 5 μM Ferrostatin-1 (HY-100579), 10 μM; deferoxamine (HY-B0988), 200 μM; necrostatin (HY-15760) 2 μM; ZVAD-FMK(HY-16658B), 5 μM were purchased from MedChemExpress (MCE). FeSO₄ 7H2O (F7002), 200 μg/mL; baicalein (465119), 20 μM were purchased from Sigma.

The osteoblastic cell line MC3T3-E1 was provided by the Cell Resource Center, Institute of Basic Medicine, Chinese Academy of Medical Sciences. Fetal bovine serum (FBS; Lonsera, Salto, Uruguay)-supplemented α-minimum essential medium (α-MEM; Biological Industries, Acre, Israel) was used to cultivate cells at 37 °C in a humidified atmosphere with 5% CO₂. Cells were cultured in the appropriate osteogenic induction medium, which contained 10 nM dexamethasone, 50 mg/L ascorbic acids, and 10 mM β-glycerophosphate (Solarbio, Beijing, China).
MC3T3-E1 cells were cultured in various groups for 72 h to test the effects of MaR1, including Normal medium (α-MEM with 5.5 mM glucose), control medium (with the addition of 20 mM mannitol and palmitate vehicle to the Normal medium of 5.5 mM glucose; Sigma, Ronkonkoma, NY, USA), T2DM medium (25 mM glucose and 200 mM sodium palmitate to mimic diabetic conditions; Alladin, Shanghai, China), and T2DM medium with MaR1 (1 or 10 nM; Cayman, Ann Arbor, MI, USA). To investigate the existence and potential mechanism of ferroptosis in osteoblasts, the ferroptosis inhibitor ferrostatin-1 (Fer-1, 5 μM; MedChemExpress, Monmouth Junction, NJ, USA) and the ferroptosis activator Erastin (Era, 1 μM; MedChemExpress, Junction, NJ, USA) were administered to the cell cultures.

Human umbilical vein endothelial cells (HUVECs) were obtained from ScienCell Research Laboratories (San Diego, CA, USA) and cultured in plates pre-coated with 0.2% gelatin in endothelial cell medium supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin, and 1% endothelial cell growth supplement (ScienCell) at 37 °C with 5% CO₂.
For each experiment using cultured HUVECs, cells were seeded in the incubator and cultured to 70–80% confluence, then exposed to L-glutamic acid (GLU, 20 mM, Cat#: G8415, Sigma-Aldrich, St. Louis, MO, USA) or N-Methyl-D-aspartic acid (NMDA, 1 mM, Cat#: HY-17551, MedChemExpress, Shanghai, China) for 24 h in the absence or presence of Ferrostatin-1 (Fer-1, 10 μM, Cat#: HY-100579, MedChemExpress), Liproxstatin-1 (Lip-1, 2 μM, Cat#: HY-12726, MedChemExpress), LB-100 (5 μM, Cat#: S7537, Sellechchem, Houston, TX, USA), Acadesine (AUCAR, 2 μM, Cat#: HY-13417, MedChemExpress), Deferoxamine (DFO, 10 μM, Cat#: HY-B0988, MedChemExpress), and Erastin (5 μM, Cat#: HY-15763, MedChemExpress) for 24 h. These plating conditions were used in all experiments, unless mentioned otherwise.

RSL3, ML210, and liproxstatin-1 (Lip-1) were purchased from Selleck Chem (Houston, TX, USA). Deferoxamine (DFO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Erastin and imidazole ketone Erastin (IKE) were purchased from MedChem Express (Monmouth Junction, NJ, USA).

Reagents: ferrostatin‐1 (CAS:347174–05‐4) and erastin (CAS:571203–78‐6) were all purchased from MedChemExpress (shanghai; China). SBFI26 (CAS:1541207–06‐0) was purchased from GLPBIO Technology (American). Antibodies: Primary antibodies were used as following: anti‐β‐actin antibody (1:1000, mAbcam 8226, Abcam), anti‐ALOX5 (1:500–1:1000, R1512‐14, HuaBio), anti‐ALOX12 (1:2000, AP8877B Abcepta Biotech), anti‐NFE2L2 (1:1000–1:2000, R1312‐8, HuaBio), anti‐GPX4 (1:500–1:2000, ET1706‐45, HuaBio), anti‐HO‐1 (1:1000, ET1604‐45, HuaBio), anti‐ATF4 (1:500–1:2000, ET1612‐37, HuaBio), secondary antibody was purchased from Beyotime (Shanghai, China).