Mc3t3 e1 murine osteoblastic cells

Cell cultures were conducted as previously described [18 (link)]. MC3T3-E1 murine osteoblastic cells and MCF-7 human breast cancer cells were purchased from the American Type Culture Collection (Manassas, VA, USA). MC3T3-E1 cells were seeded in T75 flasks, maintained at 37 °C with 5% CO₂, and cultured in the alpha-minimum essential medium (Gibco, Baithersburg, MD, USA) containing 1% penicillin/streptomycin (Gibco) and 10% fetal bovine serum (Gibco). For osteoblast differentiation, MC3T3-E1 cells were differentiated using osteoblastic differentiation medium (DM), which was prepared by adding 10 mM β-glycerophosphate and 50 μg mL⁻¹ L-ascorbic acid (Sigma-Aldrich, St. Louis, MO, USA) to the culture medium. Cells treated with DM for six days were used for ALP activity, protein, and mRNA analyses. MCF-7 cells were cultured in MEM containing 10% fetal bovine serum and 1% penicillin streptomycin in a 5% CO₂ incubator.