Sclerostin mediates inhibition of osteoblast differentiation by MDA-MB-231 cells. (A) Depletion of sclerostin from MDA-MB-231-conditioned media permits osteoblast differentiation in primary bone marrow stromal cell cultures (BMSCs). BMSCs were differentiated with normal differentiation media (Diff) or MDA-MB-231-conditioned media (MDA). Cells grown in non-differentiation media (Non-Diff) were used as negative control. MDA-MB-231 media were depleted by immunoprecipitation with a sclerostin antibody (AbScl) or a control Flag antibody (AbFlag) as indicated below the panels. (B) Alizarin red was quantified following elution and total RNAs from cells treated as in (A) 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+AbFlag by analysis of variance. (C) Sclerostin inhibits the differentiation of the pre-osteoblastic cell line MC3T3-E1. MC3T3-E1 cells were treated as in (A). (D) Alizarin red and mRNA levels of osteocalcin were determined. Data were analysed as in (B).