Figure 4

Figure 4. Pretreatment with FO restores vertebral formation in prednisolone (PDS)-post-treated zebrafish larvae. Zebrafish larvae (n = 20) at 5 days post fertilization (dpf) were treated with FO (0?100 ?g/mL) for two days, and 20 ?M PDS was post-treated for another two days. (A) At 9 dpf, zebrafish larvae were stained with 0.03% calcine and observed under fluorescence microscopy. Numbers show vertebrae (right panels) (B) Vertebral number was manually counted and indicated. (C) Relative bone area and (D) bone intensity were calculated using imageJ software and expressed. All data are presented as means ± standard error of the mean (* p < 0.05, ** p < 0.01, and *** p < 0.001). Total mRNA was extracted, and RT-PCR was performed to evaluate the gene expression of (E) osteogenic genes, including zRUNX2a, zRUNX2b, zOSX, and ALP and (F) osteoclastogenic genes such as zCTSK, zNFATc-1, zRANK, and zACP5b. ?-Actin was used as the internal control. All data are presented as means ± standard error of the mean (^# p < 0.05 and ^### p < 0.001 vs. untreated zebrafish larvae; ** p < 0.01, and *** p < 0.001 vs. PDS-treated zebrafish larvae). z: zebrafish; RUNX2a/b: runt-related transcription factor 2a/b; OSX: osterix; ALP: alkaline phosphatase; CTSK: cathepsin K; NFATc-1: nuclear factor of activated T-cells; cytoplasmic 1; RANK: receptor activator of nuclear factor ?B; and ACP5b: acid phosphatase 5b.