Bglycerophosphate bgp

Heads (calvarial bones) of 4-day-old CD-1 female mice were provided by the Biological Support Facility of the University of Manchester and Chongqing Medical University. Mice were sacrificed by cervical dislocation and dissected under sterile conditions. During dissection, the dura mater and periosteum were kept intact on the bone surfaces. The mouse calvariae were exposed as shown in The bones were cultured free-floating in 24-well tissue culture plates with DMEM (with glutamax, Invitrogen, Paisley, UK) supplemented with 10% foetal bovine serum (Invitrogen, Paisley, UK), 100 U/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Paisley, UK). One day later, the bones were randomly divided into 5 groups (n = 5/group) for micro-CT (1072, SkyScan, BE) scanning and histological staining at weekly intervals according to the scanning and staining days (Table 1). The OM was composed of DMEM supplemented with foetal bovine serum, penicillin/streptomycin, dexamethasone (Dex) (Sigma-Aldrich, Dorset, UK), ascorbic acid 2-phosphate solution (AA) (Sigma-Aldrich, Dorset, UK) and bglycerophosphate (bGP) (Sigma-Aldrich, Dorset, UK) as described in a previous article (Wu et al., 2014) . The OM-control group was scanned only at the first and final time points. The bones were cultured in a humidified atmosphere of 5% CO2 at 37 •C. The culture media were changed every 3 days.

For transient overexpression of murine AMTN, the full-length cDNA was cloned into the mammalian expression vector using the pcDNA 3.1 Directional TOPO Expression Kits (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The mouse osteogenic cell line MC3T3-E1 (subclone 14; ATCC # CRL-2594) was grown as recommended to 80% confluence in 24-well tissue culture plates, which was arbitrarily set as day 0. An amount of 1 mg of expression vector or empty vector was transfected into MC3T3-E1 cells (42) using LipofectAmine 2000 (Invitrogen) and Opti-MEM (Invitrogen). Twenty-four hours after transfection, culture medium was changed to a-MEM containing the following additives: 10 nM dexamethasone (DEX; Sigma D4902), 10 mM b-glycerophosphate (BGP; Sigma G9891), and 50 mg/mL ascorbic acid (AA; Sigma A4403). The culture medium was changed every 3 days. At the end of the culture period, cells were stained with Alizarin red and photographed. The number of calcified nodules was quantified using Image J software automated counting.
Total RNA was extracted from separate wells at day 7 and at day 14. RT-PCR for AMTN, alkaline phosphatase (ALP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; control) were then performed using SuperScript 3 reverse transcriptase (Invitrogen), advantage 2 PCR kit (Clontech, Mountain View, CA, USA), and specific primers.