Figure 7

FO enhances osteogenesis via crosstalk with the Wnt/β-catenin pathway. MC3T3-E1 cells were treated with 100 µg/mL of FO and 100 nM DEX for 7 days. (A) The cells were harvested at day 1 (D1), day 3 (D3), day 5 (D5), and day 7 (D7). Next, western blot analysis was performed to quantify β-catenin expression. (B) The cells were treated with 50 µg/mL and 100 µg/mL of FO or 100 nM DEX for 3 days. Total protein was extracted to quantify β-catenin expression in the cytosol and nucleus. (C) To evaluate the effect of β-catenin/TCF signaling, TOPFlash activity was determined. (D) Zebrafish larvae (3 dpf; n = 20) were pretreated with FH535 for 24 h and then treated with 100 µg/mL of FO or 100 nM DEX. The larvae were stained with calcein to observe vertebrae formation and mineralization 10 dpf. Relative calcein fluorescence intensity and total bone area were quantified using the Image J software and the number of vertebrae was manually counted. Significant differences among the groups were determined using the one-way ANOVA followed by Bonferroni correction. All data are presented as mean ± SEM (*** p < 0.001, ** p < 0.01, and * p < 0.05 versus untreated group). FO; fermented extract of C. gigas, DEX; dexamethasone, GP; β-glycerophosphate, dpf; days post fertilization, and UT; untreated group.