Figure 6
T63 activates canonical WNT/β-catenin pathway. (a) T63 increased TCF/LEF reporter activity at 48 h. **p < 0.01, ***p < 0.001 versus respective control (n = 3). (b) The levels of β-catenin expression in the nucleus and cytoplasm after treatment with T63 for 48 h. (c) MC3T3-E1 cells were treated with T63 (5 µM) as indicated, and the levels of phosphorylated and total GSK-3β proteins were measured. (d) DKK-1 decreased RUNX2 protein level. MC3T3-E1 cells were treated with T63 (5 μM) in the presence or absence of DKK-1 (200 ng/ml) for 48 h before western blot analysis. (e) DKK-1 decreased ALPL activity. MC3T3-E1 cells were co-treated with T63 (10 μM) and DKK-1 (200 ng/ml) for 6 days in OS medium and ALPL activity was measured. *p < 0.05, **p < 0.01 versus control, # p < 0.05 between both groups (n = 3). (f) DKK-1 impaired the mineralization. MC3T3-E1 cells were cultured in OS medium and treated as indicated for 21 days, and the quantification of calcified nodules were plotted as means ± SD (n = 3). **p < 0.01 versus control, ## p < 0.01 between both groups. Full-length blots are shown in Supplementary Fig. S5.
