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. 2006 Mar 13;172(6):909–921. doi: 10.1083/jcb.200508130

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Copyright © 2006, The Rockefeller University Press

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Figure 5. — Mdm2 is required for proper osteoblast differentiation. Calvarial osteoprogenitor cells were isolated and cultured ex vivo from E19 Col3.6-Cre–transgenic embryos bearing the R26R reporter gene, from E19 wild-type and Mdm2-conditional Col3.6-Cre–transgenic embryos. Calvarial osteoprogenitors were induced to undergo osteogenic differentiation ex vivo, resulting in proliferation (toluidine blue staining for total cell number), the formation of multilayered nodules and activation of alkaline phosphatase, and mineralization as detected by silver nitrate staining. (A) Examination R26R/Col3.6-Cre osteoprogenitor cells stained for β-galactosidase activity reveals that Mdm2 expression in Mdm2-conditional Col3.6-Cre cultures will be lost in multilayering nodules of maturing osteoblasts. (B) Upon reaching confluence, wild- type osteoprogenitor cell cultures underwent robust nodule formation and mineralization. (C) Mdm2-conditional Col3.6-Cre–transgenic cultures were unable to form a significant number of multilayered nodules, and subsequently only a small fraction of these cells were able to undergo differentiation, activate alkaline phosphatase activity, or induce mineralization. (D) Quantitative analysis by real-time PCR of the expression of early osteogenic genes type I collagen (Coll) and alkaline phosphatase (AP) and of osteocalcin (OC) at various stages of osteoblast maturation. Solid lines depict the relative transcript levels of genes in wild-type cell cultures, and dashed lines represent the levels of expression of the various genes in Mdm2-conditional Col3.6-Cre cell cultures. Expression levels of early and late osteogenic genes are reduced in Mdm2-conditional Col3.6-Cre–transgenic cells. (E) Real-time PCR analysis of Mdm2 and Runx2 expression in wild-type (WT) and Mdm2-conditional Col3.6-Cre–transgenic (MT) culture osteoblast progenitor cells during maturation. Solid and dashed lines depict the relative transcript levels of Mdm2 and Runx2 in wild-type or Mdm2-conditional Col3.6-Cre–transgenic cultures, respectively. As expected, Mdm2 levels do not increase in Mdm2-conditional Col3.6-Cre–transgenic cells during maturation. Similar induction of Runx2 and Mdm2 expression is observed in wild-type cells during maturation. (F) Western blot analysis of Runx2 protein levels in wild-type or Mdm2-conditional Col3.6-Cre–transgenic culture osteoblast progenitor cells during maturation and mineralization. Lamin-b is shown as a loading control. Decreased amounts of Runx2 protein are observed in Mdm2-conditional Col3.6-Cre–transgenic cells. (G) Quantitative PCR was performed on reverse-transcribed RNA isolated from osteoblast progenitor cells transduced with Runx2 or lacZ (negative control). Exogenous Runx2 up-regulated the expression of osteogenic maturation genes in Mdm2 mutant cells, including collagen 1, alkaline phosphatase, and osteocalcin. Error bars indicate SD.