Figure 5.

(a) C3H10T1/2 cells were infected with TAZ shRNA-producing retroviruses, and stable TAZ-depleted cells (Ti) were prepared. Retrovirus derived from a control vector was used for making control cells (Bp). The cells were induced to osteoblastic differentiation with or without Wnt3a-conditioned media. At the indicated time points, total cell lysates were prepared, and the level of TAZ was analysed by western blot. (b) Alkaline phosphatase activity was analysed by cellular staining and enzymatic assays. At 4 days after differentiation, the cells in (a) were stained with an alkaline phosphatase staining solution. For activity quantification, the differentiated cells were lysed, and the enzymatic activity of alkaline phosphatase was analysed as described in the Materials and Methods section. (c) Total RNA was isolated from the cells in (b). The expression of OC and TAZ was analysed by qRT-PCR at 2 days after differentiation