Figure 3

Runx2 binds to the SOST and CSF-2 promoter. (A) Western blot showing Runx2 expression after transfection in non-metastatic MCF-7 cells. MCF-7 cells were transfected with a Runx2-expression plasmid or the parent plasmid (-). The upper panel shows total cell lysates derived from transfections of MCF-7 cells after immunodetection with an anti-Runx2 antibody. The lower panel is a Tubulin loading control. The graphs show an increase in SOST and CSF-2 expression in non-metastatic MCF-7 cells after transfection with a Runx2-expression plasmid. (B) Diagram showing the localization of the Runx2 binding sites in the human SOST and CSF-2 promoter. The arrow indicates the start of transcription. (C) EMSA demonstrating specific binding of Runx2 to the Runx elements in the SOST and CSF-2 promoters. Nuclear extracts from MDA-MB-231 were incubated with Runx-binding sites from the human SOST or CSF-2 gene promoters. Competition assays were performed with a 100-fold molar excess of either cold wild-type (WT comp.) or mutant (Mut comp.) probes. Each gel is representative of three experiments. (D) Runx2 is recruited to the SOST and CSF-2 promoter in MDA-MB-231 cells. ChIP assays using Runx2 antibodies. Data are presented as mean ± standard deviation (S.D.) (n = 3). * indicates P < 0.05 compared with IgG by analysis of variance.