The MC3T3-E1 (mouse pre-osteoblast) cell line was obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Science and grown in α-minimal essential medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.), 100 U/ml penicillin and 0.1 mg/ml streptomycin (Gibco; Thermo Fisher Scientific, Inc.). To induce mineralization, MC3T3-E1 cells were treated for different time points (1, 3, 5, 7, 14 and 21 days) with osteogenic medium (OM), which was composed of complete medium supplemented with 50 µg/ml ascorbic acid (Sigma-Aldrich; Merck KGaA), 10 mM β-glycerophosphate (Sigma-Aldrich; Merck KGaA) and 50 ng/ml BMP2 (Novus Biologicals, LLC) in a humidified incubator at 37°C under 5% CO2. The medium was changed every 3 days. To validate the role of the PI3K/AKT signaling pathway in osteogenic differentiation, 15 mM PI3K inhibitor (LY294002; Selleck Chemicals) was added to the OM (4°C for 3 days) and the medium changed every 3 days.

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