Fig. 6: Increased mineralisation activity of Clec14a^−/−-isolated osteoblasts in vitro.

A Bone morphometric parameters in the mouse tibia of C2- and C4-treated mice. Note, C4 blocks and C2 does not block the Clec14a-Mmrn2 interaction, as previously reported²². Micro-computed tomography (μCT) analysis of trabecular percent bone volume (BV/TV%), bone surface area (B.S), trabecular number (Tb.N), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp) and trabecular extent. For BV/TV %, results are presented as median ± IQR, data was analysed with multiple Mann–Whitney tests. For the other parameters, results are presented as mean ± SEM, data was analysed with two-way ANOVA, followed by Dunnett’s post hoc, *p < 0.05. B Alpl mRNA expression was analysed by RT qPCR in calvarial osteoblasts. Results were analysed with a two-way ANOVA followed by Dunnett’s correction for multiple testing, data is presented as mean ± SD. C, D Murine calvarial osteoblasts were isolated from Clec14a^+/+ (WT) and Clec14a^−/− neonatal pups at postnatal day 4 and osteogenesis was induced with osteoinduction media in (C) freshly isolated cells prior to passage (P0) or (D) in cultured cells after passage (P1). Alpl activity was measured by ELISA after 0, 6, 8 and 10 days in culture, in Clec14a^+/+ and Clec14a^−/− osteoblasts that were incubated with either expansion media (undifferentiated – see key on graph) or osteoinduction media (differentiated – see key on graph). Data is presented as optical density (O.D.). Results were analysed with a two-way ANOVA followed by Dunnett’s post hoc, data is presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. E Representative images of alizarin red stained osteoblast-deposited calcium following 18 days in culture. F Alizarin red coverage as percentage. Results were analysed with a Mann–Whitney test, data is presented as median ± IQR, *p < 0.05. For all graphs sample size is represented by each individual dot in the graphs.