Fig. 7

Estrogen regulates vinculin expression in osteocytes and vinculin cKO mice are resistant to OVX-induced bone loss. a, b IF staining. Tibial sections of sham and OVX mice with an anti-vinculin antibody, Scale bars: 50 μm. Qauntitative data (b). n = 6 biologically independent replicates per group, **P < 0.01, versus controls, two-way ANOVA. c–e Western blot analysis. MLO-Y4 cells were treated with increasing concentration of estrogen or fulvestrant (an estrogen receptor antagonist) for 24 h. Gapdh was used as a loading control. Quantitative data from three biologically independent replicates. Results were expressed as mean ± s.d., *P < 0.05 versus control. f 3D reconstruction from μCT scans of femurs from 5-month-old control and cKO female mice performed with sham or OVX surgeries. Scale bar, 250 μm. g–i Quantitative analyses of the BMD BV/TV and Ct.Th. Results were expressed as mean ± s.d., n = 6 biologically independent replicates per group, *P < 0.05, **P < 0.01 versus controls, two-way ANOVA. j H/E staining of tibial sections. Scale bar, 200 μm. k–m Calcein double labeling. Representative images of 3-month-old male femur sections (k). Sections of non-demineralized femurs were used for measurements of MAR and BFR for the metaphyseal trabecular bones. Scale bar, 50 μm. Results were expressed as mean ± s.d., n = 6 biologically independent replicates per group, *P < 0.05 versus controls, two-way ANOVA. n–r TRAP staining. Tibial sections of 3-month-male control and cKO mice were used for TRAP staining (n). Oc.S/BS (o, p) and Oc.N/BPm (q, r) of primary and secondary cancellous bones were measured using Image-Pro Plus 7.0. Scale bar, 50 μm. Results were expressed as mean ± s.d., n = 6 biologically independent replicates per group, *P < 0.05, **P < 0.01 versus controls, two-way ANOVA