Western blot analysis
Protein lysates were generated with radio immunoprecipitation assay lysis buffer. Then protein concentrations were determined using BCA protein assay kit according to the manufacturer’s protocol. Protein samples (20 μg/lane) were loaded onto 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and electrotransferred to a polyvinylidene fluoride (PVDF) membrane. After blocking with 5% non-fat milk, membranes were incubated overnight at 4˚C with primary antibodies, including rabbit anti-rat Runx2 monoclonal antibody (1:1,000 dilution), rabbit anti-rat OPN polyclonal antibody (1:1,000 dilution), and rabbit anti-rat Wnt3a polyclonal antibody (1:400 dilution). Then, membranes were incubated with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000 dilution) for 1 h at room temperature. Membranes were visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech; Little Chalfont, U.K.). Loading differences were normalized using rabbit anti-rat GAPDH monoclonal antibody (1:2,000 dilution) or rabbit anti-rat Tubulin polyclonal antibody (1:1,000 dilution). Protein band densities on scanned films were quantified using ImageJ 1.48u software (National Institutes of Health, Bethesda, Md, USA) and compared with the control.
Analysis of mineralization
Following 14 days culture, cells were fixed, then incubated with alizarin red S as mentioned above. For further evaluation, staining was dissolved in 100 µM cetylpyridinium chloride (CPC) for 1 h at 37˚C. The optical density (OD) value of the staining dissolved CPC was measured at 562 nm using the ELISA plate reader. All experiments were repeated for three times, and each experiment in triplicate.