Fig. 6: Dmrt2 promotes Ihh expression in collaboration with Runx2.

a Primary chondrocytes were infected with the control (Cont) or Flag-tagged-Dmrt2 adenovirus, and Ihh expression was analyzed by RT-qPCR. Data are shown as fold changes normalized to Cont (mean ± s.d.) (n = 3). **p < 0.01 (vs. WT); Student’s t test. b Schematic representation of the putative Dmrt2-binding element (-BE) (GnTACA) in the mouse Ihh gene promoter. c ChIP assay using normal IgG and anti-Flag antibody. The binding of Flag-Dmrt2 to the Ihh gene promoter in primary chondrocytes was examined by PCR. d DNA pull-down assays using the Dmrt2-binding element in the mouse Ihh gene promoter. Samples precipitated with a biotin-labeled Dmrt2-BE oligonucleotide (upper panel) and cell lysates (lower panel) were examined by immunoblotting with an anti-Flag antibody. e ChIP assays using normal IgG and anti-Runx2 antibodies. The binding of Runx2 to the Ihh gene promoter in primary chondrocytes was examined by PCR. f Dmrt2 and Runx2 synergize to induce Ihh mRNA expression in primary chondrocytes. The RNA level is indicated as the fold increase compared with the level in the control. Data are shown as the mean ± s.d. (n = 3). **p < 0.01; one-way ANOVA followed by Tukey’s multiple comparison test. g Dmrt2 physically associates with Runx2. Cell lysates were immunoprecipitated with anti-Flag antibody and then immunoblotted with anti-HA antibody (top). Cell lysates were immunoblotted with anti-HA (middle) or anti-Flag (bottom) antibodies.