BMSCs, OBs and BMMs were isolated from 4 to 6 weeks old C57BL/6J, APDC-KO, and wild type (WT) mice. Isolated cells or transfected cells were plated in 10 cm dishes and maintained in αMEM supplemented with 10% FBS and 1% penicillin/streptomycin. Osteogenic differentiation of BMSCs/OBs was carried out using αMEM medium (A1049001, Glo®, MT, USA) containing 50 μg/ml ascorbic acid (Sigma-Aldrich, MO, USA), 10 mM b-glycerophosphate (Sigma-Aldrich) and 100 nM dexamethasone (Sigma-Aldrich). To promote adipogenic differentiation in BMSCs, treatment with dexamethasone (1 μM), 3-isobutyl-1-methylxanthine (IBMX) (0.5 mM), and insulin (0.5 mM) for 3 days was followed by subsequent insulin (0.5 mM) treatment. Osteoclastogenic differentiation of BMMs was processed with receptor activator of nuclear factor kappa B ligand (RANKL) (50 ng/ml) and macrophage-colony stimulating factor (M-CSF)-1 (10 ng/ml) following MSCF-1 pretreatment for 3 days. The human foetal osteoblasts (hFOBs) (Gibco™ CRL-11372™, USA) were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin with the osteogenesis-inducing medium.
Osteogenic, adipogenic and osteoclastogenic differentiation were analysed via cytological staining (Alizarin red staining for osteogenesis (Sigma-Aldrich), Oil red O staining for adipogenesis (Sigma-Aldrich), trap staining for osteoclastogenesis (Sigma-Aldrich)).
Osteogenic, adipogenic and osteoclastogenic differentiation were analysed via cytological staining (Alizarin red staining for osteogenesis (Sigma-Aldrich), Oil red O staining for adipogenesis (Sigma-Aldrich), trap staining for osteoclastogenesis (Sigma-Aldrich)).