Fig. 3

WERC stimulates osteoblast mineralization by increasing osteoblast gene expression and ERK signaling. Osteoblasts isolated from mouse calvariae were cultured in growth medium (GM, α-MEM) or differentiation medium (DM; 50 μg/mL ascorbic acid, and 10 mM β-glycerophosphate in α-MEM) in the presence of WERC (40 and 80 μg/mL). (a) Osteoblasts were subjected to ALP staining on day 5 (upper panel) or alizarin red S staining on day 10 (lower panel) (40 × magnification). Quantification of ALP activity was determined using the ALP activity assay. Quantification of mineralization was determined by measuring the amount of alizarin red S dye extracted from stained cells. (b) mRNA levels of Runx2, osterix, ATF-4, SATB2, Fra-1, JunB, collagen IIA, and osteopontin were determined by qRT-PCR on day 10. (c) After culturing osteoblasts in DM and WERC to 10 days, total cell lysate (30 μg) was extracted and subjected to western blot analysis with antibodies specific to ERK, p38, and β-actin. *p < 0.05, **p < 0.01, ***p < 0.001