DKK1 knockdown suppresses matrix mineralization of osteoblast differentiation. (A) For knockdown of DKK1, cells were transfected by shRNA and then treated with doxycycline 0, 2, 5 μg/ml for 48 h. The treated cells were analyzed by RT-PCR (left) and immunoblotting (right). (B-E) The DKK1 knockdown cells were stimulated with osteogenic media containing doxycycline 5 μg/ml during indicated days. Osteogenic differentiation was assessed by (B) ARS staining (left), Von kossa staining (center) and HA staining (right); scale bar is 200 μm. (C) Quantification of (B). (D, E) stable DKK1 knockdown cells were treated with osteogenic media containing doxycycline 5 μg/ml. (D) After 7 days, the cells were fractionated into cytosol and nucleus proteins and analyzed by immunoblotting. (E) At indicated days, cells were analyzed by RT-qPCR for DKK1, RUNX2, and OCN mRNA levels. Doxy, doxycycline; Days, osteogenic days; ARS, alizarin red s; HA, hydroxyapatite; BF, bright field. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant.
