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.
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.