Figure 4.

SOX9 expression was significantly decreased in TMJ cartilage of 3-month old β-cat(ex3)^Agc1CreER and immortalized TMJ fibrocartilage cells with BIO treatment. (A)β-cat(ex3)^Agc1CreER mice were generated by crossing Agc1-CreER^T2 mice with β-catenin(ex3)^floxflox mice. TMJ sample were harvested from 3-month old mice after they were injected with tamoxifen at the age of 2 weeks for 5 consecutive days. Immunohistochemical (IHC) analysis showed that β-catenin was highly expressed in TMJ cartilage of β-cat(ex3)^Agc1CreER mice compared with Cre negative mice (green arrows) but SOX9 expression was reduced in TMJ cartilage of β-cat(ex3)^Agc1CreER mice in TMJ cartilage of β-cat(ex3)^Agc1CreER mice (red arrows). (B) The protein level of SOX9 were analyzed on cell lysates from immortalized TMJ fibrocartilage cells with the indicated treatment for 24 h by western blotting. Data illustrate that activation of canonical WNT-signaling by BIO significantly downregulated the expression of SOX9 but TGF-β or BMP2 highly upregulated the expression of SOX9. Equal sample loading was also confirmed by detecting β-actin. Data for a typical experiment are presented and experiments were performed more than three times from different passages with similar results.