DKK1 overexpression induces non-canonical WNT signaling during osteoblast differentiation. (A) Osteoprogenitor cells were treated DKK1 for 24 h and transfected with Empty and DKK1 plasmid DNA for 48 h. The cells were analyzed by RNA sequencing, and screened by DKKs, CAMKs, CREBs and WNTs. (B) Cells were treated DKK1 and verapamil in dose-dependent manner. DKK1 was treated for 1 h, verapamil was pre-treated 10 min before DKK1 treatment. The cells were measured intracellular Ca+ influx. (C-E) Osteoprogenitor cells were transfected by Empty and DKK1 plasmid DNA, and the cells were stimulated by osteogenic differentiation. (C) At indicated days, the cells were analyzed by immunoblotting. (D, E) Osteogenic differentiation for 14 days. (D) The cells were harvested and fractionated into cytosol and nucleus proteins. The proteins were analyzed by immunoblotting. (E) The cells were subjected to immunofluorescence; DKK1 (green), p-CREB1 (red) and DAPI (blue); scale bar is 50 μm. over., overexpression; treat., treatment; Ca+, calcium; OM days, osteogenic days. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.