Figure 1

Runx2 deficient mice have higher levels of BMP-3B. (a) Total RNA from mesenchymal cells of calvarial tissue from wild type and Runx2^-/- (17.5 dpc) was hybridized with osteogenesis-related cDNA array. Signal of BMP-3B on blots is indicated by an arrow. Control genes (GAPDH, cyclophilin A) are shown in lower panel. (b) BMP-3B mRNA levels (normalized to GAPDH) in calvarial mesenchymal cells from wild type (WT) and Runx2^-/- animals as detected by qRT-PCR analysis. (c) Primary calvarial cells from Runx2 deficient animals were transduced with empty vector (EV) or Runx2 expressing adenovirus with increasing multiplicity of infection (MOI 10 and 20) for 48 hr. Total RNA isolated from the infected cells was utilized to examine BMP-3B mRNA as detected by qRT-PCR analysis. (d) Runx2 mRNA expression levels were examined by qRT-PCR and the gene expression levels were normalized to 28S internal control. The gene expression levels were calibrated to IMR-90 cells. (e) BMP-3B mRNA expression levels were examined by qRT-PCR and the gene expression levels were normalized to 28S internal control. The BMP-3B gene expression levels were calibrated to Runx2 levels in IMR-90 cells. (f) Runx2 protein levels were examined in nuclear extracts of normal lung fibroblast cells (IMR-90 and WI-38) and lung cancer cell lines (H720 and H1299) by immunoblotting with Runx2 monoclonal antibody. Runx2 expression levels were normalized to internal control LaminA/C protein. (g) Runx2 intracellular localization was determined by immunofluorescence of endogenous Runx2 protein in WI-38 and H1299 cells. The punctate (Alexa 488, green) signal shows Runx2 staining while nuclei are revealed by 4, 6-diamidino-2-phenylindole (DAPI, blue) staining.