Figure 5: Col10a1 is the direct target of Foxc1 in vitro and in vivo.

(a) Effects of Foxc1 on hypertrophic chondrocyte gene expression. Primary chondrocytes were infected with control or Foxc1 adenovirus and chondrocyte gene expression was analysed by RT–qPCR. Data are shown as the mean±s.d. (n=3). (b) Schematic presentation of the putative Foxc1-binding element (ATTTATTTA) in the promoter region of mouse Col10a1. (c) DNA pull-down assays using the Foxc1-binding element in the Col10a1 promoter. 293FT cell lysates precipitated with biotin-labelled Foxc1-BE oligonucleotide (upper) and total cell lysates (lower) were assessed by immunoblotting with an anti-Flag antibody. (d) ChIP assays using anti-Foxc1 antibody. The binding of Foxc1 to the Col10a1 promoter was examined by qPCR. Data are shown as the mean±s.d. (n=6). **P<0.01 (versus Control); Student’s t-test. (e) COS-7 cells were transfected with reporter constructs containing 6xFoxc1-BE from the Col10a1 gene promoter (upper panel) together with an empty vector and Foxc1. Data are expressed in relative luciferase units (mean±s.d., n=3). **P<0.01 (versus 0 ng); one-way analysis of variance (ANOVA) followed by the Tukey–Kramer test. (f) Immunofluorescence analysis of tibial growth plate chondrocytes in newborn WT and Foxc1^ch/ch littermates. Paraffin sections of tibia were subjected to haematoxylin and eosin (H&E) staining and immunofluorescence analyses using anti-Col2 and anti-Col10 antibodies. Scale bar, 200 μm. (g) Quantitative analysis of the lengths of Col2-positive and Col10-positive zones. Data are shown as the mean±s.d. (n=5) **P<0.01 (versus WT); Student’s t-test. (h) Immunofluorescence analysis of sternums in newborn WT and Foxc1^ch/ch mice. Paraffin sections of sternums of the mice were subjected to immunofluorescence analyses using an anti-Col2 or anti-Col10 antibody. The H&E staining images are higher magnification images of those presented in Fig. 2b. Scale bar, 100 μm. (i) Primary chondrocytes from E18.5 WT and Foxc1^ch/ch littermates were infected with control (Cont) or Foxc1 adenovirus as indicated. Col10a1 mRNA expression was determined by RT–qPCR. Data are shown as the mean±s.d. (n=3) **P<0.01, *P<0.05; one-way ANOVA followed by the Tukey–Kramer test.