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. 2004 May 1;18(9):1072–1087. doi: 10.1101/gad.1171104

Figure 6.

Figure 6.

Inhibition of cell proliferation and Cyclin D1 expression in Col2a1/Sox9 knock-in mutant embryos, and negative functional interactions between Sox9 and β-catenin in vitro. (A) PCNA staining shows a decrease in PCNA-positive cells (brown nuclei) in the radius of E16.5 mutant embryos. The double arrows indicate the zone of proliferating chondrocytes. Boxed regions show a higher magnification of proliferating chondrocytes. BrdU incorporation also decreases in the radius of E16.5 mutant embryos. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t-test. (*) Statistically significant difference between wild-type and mutant embryos at p < 0.001. (B) Expression of Cyclin D1 mRNA (in situ hybridization; ISH) and protein (immunohistochemistry; IHC) in the radius of E16.5 wild-type and mutant embryos. The arrows indicate Cyclin D1-positive proliferating chondrocytes. (C) Activation of the Cyclin D1 promoter by stβ-catenin is inhibited by Sox9 in 293 cells. Cells are cotransfected with 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9. Activity of the Cyclin D1 promoter is measured by luciferase assay. Cell extracts are fractionated by electrophoresis and blotted with anti-myc and anti-HA antibodies. Cotransfection of Sox9 and β-catenin results in a reduction of the levels of both proteins. (D) Activation of TOPFLASH by stβ-catenin is inhibited by Sox9. TOPFLASH and FOPFLASH activities are measured by luciferase assay. Cotransfection of 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9 in 293 cells results in a reduction of the levels of these proteins in a dose-dependent manner. (E) Activation of the Col2a1/4 × 48-bp enhancer reporter construct was inhibited by stβ-catenin in C3H10T1/2 cells. Cotransfection of 6x myc-tagged stβ-catenin and 3xHA-tagged Sox9 results in a reduction of the levels of these proteins in a dose-dependent manner. Statistical significance is assessed by one-way analysis of variance and unpaired Student's t-test. (*) Statistically significant difference between Sox9 transfection and Sox9 and stβ-catenin cotransfection at p < 0.001.