Fig. 2

EGFL6 treatment enhances osteogenic differentiation of rat bone marrow mesenchymal stem cells (BMSCs) in vitro. a BMSC viability after treatment with different concentrations of EGFL6, as assessed by the CCK-8 assay. Values are means±SD. b Images of Alizarin Red S (AR-S)-stained BMSCs treated with EGFL6, showing increased mineralization (rust-colored deposits). Osteogenic differentiation of BMSCs was examined on day 14. c Images of alkaline phosphatase (ALP)-stained BMSCs treated with EGFL6. Osteogenic differentiation of BMSCs was examined on day 3. Insets in b and c show low-magnification images of entire culture well. Scale bars, 250 μm. BMSCs were treated with different concentrations of EGFL6 for 5 or 10 days. d Expression levels of angiogenesis- and osteogenesis-related markers in BMSCs following treatment with/without EGFL6 for 5 days, as evaluated by RT-PCR. The housekeeping gene GAPDH served as an internal control. e Western blots of lysates from cultured BMSCs treated with/without EGFL6 for 5 or 10 days. Blots were probed with antibodies against different markers for angiogenesis (VEGF-A), osteogenesis (BMP2, CXCR4, RUNX2), and the Wnt/b-catenin signaling pathway (b-catenin, pb-catenin, active β-catenin, and pGSK3β). f Quantitation of expression of angiogenesis-, osteogenesis-, and pathway-related marker proteins in panel e. Significant differences among groups were determined by one-way ANOVA and post hoc Dunnett’s test; *p < 0.05; **p < 0.01; and ***p < 0.001