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. 2007 Jul;18(7):2411–2418. doi: 10.1091/mbc.E06-08-0743

Figure 6.

Figure 6.

Regulation of Runx2 by Gli2 during osteoblast differentiation. (A) C3H10T1/2 cells infected with control, Myc-tagged Gli2, or Flag-tagged Gli3 adenovirus were incubated in the presence or absence of Ihh (500 ng/ml) or BMP2 (300 ng/ml) for 7 d. The lysates of these cells were analyzed by immunoblotting with anti-Runx2 (top) or β-actin (bottom) antibody. Similar results were obtained from three independent experiments. (B) C3H10T1/2 cells were infected with control or Myc-tagged dominant-negative Gli2 adenovirus (DN-Gli2) and incubated in the presence or absence of Ihh (500 ng/ml) for 7 d. The lysates of these cells were analyzed by immunoblotting with anti-Runx2 (top), β-actin (middle), or Myc (bottom) antibody. Similar results were obtained from three independent experiments. (C) C3H10T1/2 cells were infected with control, Myc-tagged Gli2, or Runx2 adenovirus and cultured for 7 d. The cells were then analyzed for ALP activity. Data represent mean ± SD. *p < 0.05 (vs. control); #p < 0.01 (vs. control); $p < 0.05 (vs. Runx2, Gli2). Similar results were obtained from three independent experiments. (D) C3H10T1/2 cells were transfected with osteocalcin luciferase and TK-renilla reporter constructs together with pcDNA3 (Cont), Myc-tagged Gli2 expression vector, Runx2 expression vector, or both for 2 d. Luciferase activity in the lysates was measured. Data represent mean ± SD. *p < 0.05 (vs. control); #p < 0.05 (vs. Runx2, Gli2). Similar results were obtained from three independent experiments. (E) C3H10T1/2 cells were infected with Gli2 and/or Runx2 adenovirus and incubated for 7 d. The amount of osteocalcin in the conditioned medium was determined using a mouse osteocalcin EIA kit. *p < 0.05 (vs. control); #p < 0.01 (vs. control); $p < 0.01 (vs. Runx2, Gli2). Similar results were obtained from three independent experiments. (F) C3H10T1/2 cells were transfected with Myc-tagged Gli2 expression vector, Flag-tagged Runx2 expression vector, or both and incubated for 2 d. The lysates of these cells were immunoprecipitated with anti-Flag antibody and immunoblotted with anti-Myc antibody (top). The expression levels of Myc-tagged Gli2 or Flag-tagged Runx2 were monitored by immunoblotting with anti-Myc (middle) or anti-Flag (bottom) antibody. Similar results were obtained from three independent experiments. (G) C3H10T1/2 cells were infected with control, Flag-tagged Gli3, or Runx2 adenovirus and cultured for 7 d. The cells were then analyzed for ALP activity. Data represent mean ± SD. *p < 0.05 (vs. control). Similar results were obtained from three independent experiments.