Figure 2.

MKs promote OBs proliferation and differentiation, as well as ECs tube formation. (A) Representative immunostaining images of osteocalcin⁺ cells (arrowheads) on the surfaces of TB and EB from MK^deleted mice and their littermate controls (n=6 mice per group). The quantification of osteocalcin⁺ cells are shown in the right panel. Scale bar, 100 µm. (B) The concentration of osteocalcin in the BM and serum of MK^deleted mice and their littermate controls, determined by ELISA (n=6 mice per group). (C) Proliferation of OBs in direct culture with MKs for 5 days (n=6 per group). (D) Pre-OBs were induced differentiation into mature OBs in osteogenic differentiation medium without or with MKs-CM from WT mice after 7 days. Representative alkaline phosphatase staining images (left) and quantification of the activity of alkaline phosphatase was calculated (right) (n=6 per group). (E) Pre-OBs were induced differentiation into mature OBs in osteogenic differentiation medium without or with MKs-CM from WT mice after 21 days. Representative Alizarin red staining images (left) and quantification of matrix mineralization was calculated (right) (n=6 per group). (F) Representative immunostaining images of CD31 (red) and Emcn (green) in the BM (left) and endocortical bone surfaces (right) of MK^deleted mice and their littermate controls. The quantification of CD31^hi Emcn^hi (yellow) cells are shown in the right panel (n=6 mice per group). Scale bar, 100 µm. (G) Representative images of tube formation of ECs (with or without co-cultures of OBs) after addition of MKs-CM. The quantitative analysis of cumulative tube length is shown in the right panel (n=6 per group). Scale bar, 100 µm. Data are shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. For all panels in this figure, data are representative of three independent experiments.