Figure 3. Runx2 regulates RANKL transcription.

(A) Putative transcription factors which bind to 5'-flanking and promoter regions of murine RANKL (AF332141) were identified through the TFSEARCH engine (http://www.cbrc.jp/research/db/TFSEARCH.html). Three Runx2-binding sites were identified within the −400 to −200 bp region and one additional binding site was found near −200 bp. The putative Runx2 binding sites (Runx2-1, Runx2-2, Runx2-3, and Runx2-4) and primers for ChIP assay are indicated. (B) ChIP assay with Runx2 immunoprecipitates (anti-Runx2; Santa Cruz) and PCR using primer sets for whole, R1&2, and R3&4. Representative pictures of 3 independent experiments are shown. (C) ChIP assay with PCR primer sets R1, R2, R3, and R4. Representative pictures of 3 independent experiments are shown. (D) EMSA was performed using four probes (P1, P2, P3, and P4) containing the putative Runx2 binding elements. Probes carrying mutations of the Runx2-binding sites (P1m, P2m, P3m, and P4m) were used as negative controls. Representative EMSA pictures of 3 independent experiments are shown. (E) Competitive EMSA was performed using 100-fold molar excess amounts of unlabeled probes. Probes carrying mutation of the Runx2-binding sites were used as negative controls. Representative EMSA pictures of three independent experiments are shown. (F) Specific antibody for Runx2 was used to detect supershift in Runx2 binding complex. A representative EMSA picture of 2 independent experiments are shown. (G) VSMC with stable Runx2 knockdown by shRNA against Runx2 (Lenti-shRunx2) or control lentiviruses encoding GFP (Lenti-GFP) were exposed to 0.4 mM H₂O₂ for 2 weeks. Expression of Runx2 protein was determined by Western blot analysis. A representative blot of 2 independent experiments is shown. (H) Expression of RANKL transcript in control or Runx2 knockdown VSMC in G was determined by RT-PCR. Representative results from two independent experiments are shown. (I) VSMC were transduced with Ad-Runx2 or control virus (Ad-GFP) and cultured in osteogenic media for 2 weeks. Expression of RANKL transcript was determined by RT-PCR. Representative RT-PCR results of 2 independent experiments are shown.