Fig. 2: Enhanced OC differentiation and bone resorption by 4T1 BC-cell OC interaction is mediated by Mstn in vitro.

A Mstn expression in naïve (healthy control) and in tibiae 14 days after 4T1-luc2 tumor cell injection. E: epiphysis, GP: growth plate, TB: trabecular bone, BM: bone marrow. B Representative Western Blot of Mstn in 4T1, MDA-MB-231 breast cancer cell lines. Positive controls: pos. ctrl1: osteocyte cell line MLO-Y4, pos. ctrl2: mouse skeletal muscle tissue lysate. C OC differentiation in co-cultures of WT BMDMs and 4T1 tumor cells (n = 6) and anti-myostatin antibody (5 µg/ml). D OC differentiation of WT BMDMs cultured with 10% conditioned medium of 4T1 tumor cells and anti-myostatin antibody (n = 8). C, D TRAP staining was used to visualize mature OCs. E WT BMDMs were co-cultured with 4T1 tumor cells on calcium phosphate coated plates and stimulated with anti-Mstn antibody (5 µg/ml) (n = 8). F WT BMDMs were cultured on calcium phosphate coated plates and treated with 10% conditioned medium of 4T1 tumor cells and 5 µg/ml anti-Mstn (n = 8). C–F All cells were cultivated in the presence of 30 ng/ml M-CSF and 50 ng/ml RANKL. Control: differentiated WT BMDMs only. All data are mean ± SEM, one-way ANOVA, Kruskal-Wallis test with multiple comparisons, *p ≤ 0.05, **p ≤ 0.01; scale bar 200 μm.