Runx2 expression in MDA-MB-231 cells is required for inhibition of osteoblast differentiation. (A) Western blot showing stable MDA-MB-231 cells knocked down for Runx2 or CBFβ. The left panel shows total cell lysates derived from stables MDA-MB-231 cells, transfected with a shRNA non-specific (shNS) or shRNA for Runx2 (shRunx2) or CBFβ (shCBFβ). The lower panel is a Tubulin loading control. Graphs show that expression SOST is significantly reduced in the cell lines. Data are presented as mean ± standard deviation (SD) (n = 3). Statistical evaluation of significant differences was performed using the Student's t-test. Asterisk (*) indicates P < 0.05 when compared to control (shNS). (B) Knock-down of Runx2 in MDA-MB-231 cells permits the differentiation of osteoblasts. BMSCs were differentiated with normal differentiation media (Diff), conditioned media from stable shNS cells (MDAshNS) or shRunx2 cells (MDAshRunx2). Cells grown in non-differentiation media (Non-diff) were used as negative control of differentiation. (C) Alizarin red was quantified following elution and total RNAs from cells treated as in (B) were used to analyse expression of the osteoblast differentiation marker, osteocalcin. Data are presented as mean ± standard deviation (SD) (n = 3). One-way analysis of variance was used and multiple comparisons between individual groups were assessed by the method of Tukey. * indicates P < 0.05 compared to Non-Diff by analysis of variance; # indicates P < 0.05 compared to Diff+MDA shNS by analysis of variance. (D) Diagram depicting the proposed role of Runx2 in the modulation of bone cell functions by regulating secreted factors. Other genes involved in invasion are omitted for clarity.