Figure 1.

(A) mRNA levels of hypertrophic and terminal differentiation markers, including ANK, APase, MMP-13, osteocalcin (OC), runx2 and type X collagen α1(X)), and type II collagen (α1(II)), and (B) mineralization of growth plate chondrocytes cultured in the presence of various concentrations of extracellular P_i(1, 2.5, 4, and 8mM) and in the absence (−PFA) or the presence of PFA (+PFA). (A) The levels of these hypertrophic and terminal differentiation marker and type II collagen mRNAs were determined after 2-day treatment with P_i by real-time PCR and SYBR Green and normalized to the 18S RNA levels. Data are means of triplicate PCRs using RNA from three different cultures; error bars represent standard deviations. (B) The degree of mineralization of growth plate chondrocyte cultures treated for 4 days with various concentrations of P_i(1, 2.5, 4mM) in the absence (−RA) or presence of 35nM RA (+RA) was determined using alizarin red S staining. To quantitate the alizarin red S stain each dish was incubated with cetylpyridinium chloride for 1h. The optical density of alizarin red S stain released into solution was measured at 570 nm, and normalized to the total amount of protein. Data are means of four experiments; error bars represent standard deviations.