FIGURE 7.

Disruption of RUNX2-SMAD interaction results in altered expression of osteoblast markers genes. The osteogenic capacity of the wild-type and mutant Runx2 proteins were tested in a reconstitution assay of Runx2^-/- cell line. Cells were plated in 6-well dishes, and upon confluency infected with Runx2 adenovirus for 4 h. Cells were then washed and cultured for indicated days in osteogenic media (αMEM supplemented with 10% fetal bovine serum, 10 mm β-glycerol phosphate, and 50 μg/ml ascorbic acid). Control and Runx2-infected cells were harvested for RNA isolation at indicated days. Real-time reverse transcription-PCR analyses were performed to assess expression of the cell cycle, RUNX2 target, and other transcription factor genes during osteoblastic differentiation. Data are presented as relative transcript normalized to glyceraldehyde-3-phosphate dehydrogenase levels in the same sample. All experiments were performed in duplicates, and mean normalized values with standard deviation are shown.