Induction of osteogenic, chondrogenic, and adipogenic lineage markers in piMEFs <i>in vitro</i>.

<p>(<b>A</b>) Expression of lineage-specific regulators in piMEFs stimulated by BMP9. Subconfluent piMEF cells were infected with AdBMP9 or AdGFP. Total RNA was isolated at the indicated time points and subjected to RT-PCR reactions. The cDNA products were used as templates for semi-quantitative amplification of mouse Runx2, Sox9 and PPARγ2 transcripts. All samples were normalized with GAPDH expression levels. (<b>B</b>) Induction of early osteogenic marker alkaline phosphatase (ALP) in primary MEFs and piMEFs. Subconfluent primary MEFs and piMEFs were infected with AdBMP9 or AdGFP. ALP activity was quantitatively determined at days 3, 5 and 7. (<b>C</b>) Matrix mineralization assessed with Alizarin Red S staining. Subconfluent MEFs and piMEFs were infected with AdBMP9 or AdGFP and maintained in mineralization medium for 14 days. Cells were fixed and stained with Alizarin Red S. (<b>D</b>) Adipogenic differentiation assessed with Oil Red O staining. Subconfluent MEFs and piMEFs were infected with AdBMP9 or AdGFP for 10 days. Cells were fixed and stained with Oil Red O staining. Each assay condition was done in triplicate. The assays were repeated in at least two independent batches. Representative results are shown.