Retinoic Acids Potentiate BMP9-Induced Osteogenic Differentiation of Mesenchymal Progenitor Cells
Figure 3
Retinoids potentiate BMP9-induced late osteogenic markers and matrix mineralization in MPCs.
(A) qPCR analysis of retinoic acids and BMP9 induced osteopontin (OPN) expression. Subconfluent MEFs were infected with AdBMP9 or AdGFP (i.e., -BMP9 groups, MOI = 5), and then treated with 9CRA (20 µM), ATRA (20 µM), or solvent control. At day 7 and day 9, the cells were collected for total RNA isolation. RNA was subjected to RT-PCR transcription, which was used as templates for qPCR analysis using primers specific for mouse OPN. Each assay condition was carried out in triplicate. All samples were normalized using endogenous levels of GAPDH. “*”, p<0.05; “**”, p<0.01 (vs. control groups). (B) qPCR analysis of retinoic acids and BMP9 induced osteocalcin (OC) expression. Samples prepared in (A) were used for qPCR analysis using primers specific for mouse OC. Each assay condition was carried out in triplicate. “*”, p<0.05; “**”, p<0.01 (vs. control groups). (C) Western blotting analysis of retinoic acids and BMP9 induced OPN and OC expression. MEFs were infected with AdBMP9 or AdGFP (i.e., -BMP9 groups, MOI = 5), and then treated with 9CRA (20 µM), ATRA (20 µM), or solvent control. At day 7, cells were lysed and subjected to Western blotting analysis using anti-OPN or anti-OC antibody (Santa Cruz Biotechnology). Anti-β actin antibody was used to demonstrate equal loading of all samples. (D) Retinoic acids and BMP9 induce matrix mineralization. MEFs were infected with AdBMP9 or AdGFP (i.e., -BMP9 groups, MOI = 5), and then treated with 9CRA (20 µM), ATRA (20 µM), or solvent control. At day 14, cells were subjected to Alizarin Red S staining. Experiments were carried out in duplicate and representative results are shown.