Fig. 4

Dickkopf-related protein 1 (DKK1) partially suppresses EGFL6-enhanced BMSC osteogenesis in vitro. a Light micrographs of ALP-stained BMSC cultures on day 3 of differentiation. BMSCs were treated with 200 ng/ml EGFL6 to enhance osteogenic differentiation, and then supplemented with/without 0.3 μg/ml DKK1. Insets show low-magnification images of entire culture well. Scale bars, 250 μm. b AR-S staining of differentiated BMSCs showing mineralization (red) on day 14 after DKK1 application. Insets show low-magnification images of entire culture well. Scale bars, 250 μm. c Western blots showing the expression of osteogenic-specific and Wnt/β-catenin signaling-related proteins in BMSCs treated with/without EGFL6 and with/without DKK1. GADPH is the loading control. d Quantitation of osteogenic-specific and Wnt/β-catenin signaling-related proteins normalized to control condition (NS; black-colored bars). e, f, g Immunofluorescent images of BMSCs stained for RUNX2 (green) or active β-catenin (green). BMSCs were cultured with 200 ng/ml EGFL6 to enhance BMSC osteogenesis, and then treated with/without 0.3 μg/ml DKK1, an antagonist of Wnt/β-catenin signaling. Scale bar, 100 μm. Quantitation of RUNX2 or active β-catenin immunofluorescent staining showing mean relative fluorescence of DKK1 + EGFL6 (blue-colored bars) and EGFL6 alone (pink-colored bars) conditions normalized to control fluorescence (no DKK1, no EGFL6; gray-colored bars). Significant differences were evaluated by one-way ANOVA and post hoc Dunnett’s tests for all panels; *p < 0.05; **p < 0.01; and ***p < 0.001