Figure 3.

Baf45a functions promote odontoblast differentiation. (A) Relative expression levels of Baf45a were obtained by real-time RT-qPCR using total RNA isolated from murine odontoblast OD-21 cells induced to differentiate for 20 days. β-Actin cDNA was used as a loading control. (B) Real-time RT-qPCR analysis for the relative expression levels of Dspp using total RNA isolated from murine odontoblast OD-21 cells differentiated for 20 days. (C) Representative of mRNA analysis in triplicate (n=3) of Baf45 homologs in OD-21 cells induced to differentiate for 20 days. (D) Baf45a and Baf45d responsiveness to BMP2 and TGFβ in OD-21 odontoblast cells. RT-qPCR analysis from OD-21 cells treated with BMP2 (100 ng/ml) and TGFβ (10 nM) at 80-90% confluence of culture and harvested 24 h post-treatment. (E) OD-21 cells were treated without shRNA (WT), control shRNA (con sh), and shRNA specific to Baf45a (sh Baf45a). After 72 hrs. post-treatment, RT-qPCR was performed on the RNA isolated from the harvested cells to assay the expression of Baf45a. Gapdh expression was used to normalize the relative gene expression. (F) Effect of Baf45a shRNA was assayed on the expression of Atf4, Klf4, and Runx2. (G) odontoblast differentiation markers Dmp1 and Dspp1 were assayed. Gapdh expression was assessed to normalize the relative expression. Statistical significance was determined by Student’s t-test (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 versus matched control).