Fig. 2.

Extracellular S100A4 inhibited matrix mineralization. (A) Mouse calvarial preosteoblasts cultured with osteogenic medium containing vehicle (V) or indicated concentration of recombinant mouse S100A4 for the indicated days were subjected to viability assay. (B) Mouse calvarial preosteoblasts were cultured with vehicle (Veh.) or S100A4 (1 μg/ml) in an osteogenic medium for 10 days. Alizarin-red staining was performed, and a representative image is shown. (C) The intensity of the image from (B) was measured using ImageJ program (left). The intensity of Alizarin-red stain solubilized with cetylpyridium chloride was also quantified (right). (D) Mouse calvarial preosteoblasts were cultured with a vehicle or S100A4 (1 μg/ml) in osteogenic medium for the indicated days and subjected to real-time PCR for Bglap (osteocalcin) expression. HPRT was used as an internal control. (E) Mouse calvarial preosteoblasts were cultured with a vehicle or S100A4 (1 μg/ml) in osteogenic medium for the indicated days. The osterix protein levels were assessed by Western blotting and β-Actin was included as a loading control. Error bars represent the SD of mean values. **P < 0.01 versus Veh. n.s.: not significant.