FIG. 2.

The effect of TSH on osteoclast differentiation. ES cells were cultured with the four ODFs for 12 days, allowing the formation of osteoclasts in the absence or presence of increasing concentrations of TSH as indicated. (A) TSH decreased the number of TRAP-positive multi-nucleated cells. TRAP-positive multi-nucleated cells containing three or more nuclei were counted. Three wells were counted in total for each condition and results expressed as mean ± SEM of triplicate cultures. (B) TSH decreased specific osteoclast markers in cultured cells. The cells were subjected to real-time qPCR analysis of TRAP, CTR, CTSK, MMP-9, and CA2 gene expression. Values were normalized to GAPDH RNA expression. TSH showed a dose-dependent decrease in all osteoclast markers. The results are representative of three independent experiments. *The p-value comparing ODFs treatment versus ODFs treatment in the presence of TSH was < 0.01. TSH, thyroid-stimulating hormone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.