Fig 2: Effects of AZD4547 on expression of RANKL, M-CSF, and osteocalcin following FGFR activation induced by FGF-1 and MDA-MB-134-VI supernatant. Effects of AZD4547 on FGF1-stimulated (a) and MDA-MB-134-VI supernatant-stimulated (b) expression of osteoblast-related genes (Tnfsf11 and Csf1) in pre-osteoblast cells were examined by quantitative RT-PCR. Cells were stimulated with FGF1 (10 ng/mL) or MDA-MB-134-VI supernatant (diluted 1:20), and then treated with AZD4547 (0.2 nM) for 2 h before total RNA lysates were prepared. Each data point represents the mean value acquired from 3 experiments with the standard error. DihydroxyvitaminD3 is mean as positive control of osteoblasts. C.M, MDA-MB-134-VI supernatant. ***p < 0.001, in one-way ANOVA.

Fig 3: Protein expression levels of RANKL induced by FGF-1 or MDA-MB-134-VI supernatants are reduced by AZD4547. The effects of AZD4547 on FGF1 (10 ng/mL)-induced (a) and MDA-MB-134-VI supernatants (1:20)-induced (b) RANKL protein expression was examined by western blot analysis after treatment with AZD4547 (0.2 nM). Expression was evaluated by quantitative densitometry using QuantityONE v461. Each point represents the mean of 3 independent determinations with the standard error. C.M, MDA-MB-134-VI supernatant. *p < 0.05, **p < 0.01, ***p < 0.001 in one-way ANOVA.

Fig 4: Evaluation of FGFR activation in pre-osteoblast cells induced by MDA-MB-134-VI cell supernatant. The changes in FGFR phosphorylation levels after stimulation with FGF1 (a) and MDA-MB-134-VI supernatants (b) were investigated by western blot analysis. The cells were pre-stimulated with FGF1 and MDA-MB-134-VI supernatants under serum-free conditions for 24 h and incubated for the indicated times in the presence or absence of FGF1 and MDA-MB-134-VI supernatants. β-Actin was used as a loading control. C.M, MDA-MB-134-VI supernatant.