DKK1 overexpression promotes matrix mineralization of osteoblast differentiation. (A-F) Osteoprogenitor cells were transfected with Empty and DKK1 Plasmid DNA. (A) After transfection for 2 days, overexpression of DKK1 was confirmed by RT-PCR (left), RT-qPCR (center) and immunoblotting (right). (B-F) Transfected cells were stimulated with osteogenic differentiation. At the indicated days, osteogenic differentiation was evaluated by (B) ALP staining (left), ALP activity (center) and Collagen staining (right); scale bar is 200 μm, (C) ARS staining (left), Von kossa staining (center) and HA staining (right); scale bar is 200 μm. (D) Quantification of (C). (E) At the indicated days, cells were analyzed by RT-qPCR with DKK1, RUNX2, and OCN and normalized to GAPDH. (F) Transfected cells were stimulated with osteogenic differentiation for 14 days and immunostaining with DKK1 (green), OCN (red), and DAPI (blue) were analyzed; scale bar is 50 μm. (G) SaOS2 cells were transfected with ALP, OCN, or OSE promoter plasmids for 24 h, treated with DKK1 dose-dependent manner for 24 h, and analyzed with a luciferase assay. over., overexpression; OM days, osteogenic days; ALP, alkaline phosphatase; ARS, alizarin red s; HA, hydroxyapatite; BF, bright field. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
