Figure 5.

Met and NF-κB inhibitors suppress HGF-induced RANKL expression. (A) ST2 cells, MC3T3-E1 cells, and mouse BMSCs were incubated in 96-well plates for 24 h and then treated with various concentrations of tepotinib, bortezomib, or DMF. After 5 days, cell viability was quantified by the trypan blue dye assays. The results are representative of five independent experiments. * p < 0.01 vs. the controls (one-way ANOVA with Dunnet’s test). (B–D) ST2 cells, MC3T3-E1 cells, and mouse BMSCs were treated with 1 μM tepotinib, 5 nM bortezomib, or 10 μM DMF. After incubation for 72 h, HGF was added to give the final concentration of 10 ng/mL. (B) After incubation for 4 h, RNA was extracted, and RANKL mRNA expression was analyzed by real time PCR. The results are representative of five independent experiments. * p < 0.01, as compared to controls (one-way ANOVA with Dunnett’s test). (C) After incubation for 72 h, the cell lysates were analyzed by western blotting using anti-RANKL antibody. (D) Quantification of the amount of RANKL, normalized to the amounts of β-actin. The results are representative of five independent experiments. * p < 0.01, compared to controls (one-way ANOVA with Dunnett’s test). (E) After culturing ST2 cells, MC3T3-E1 cells, and mouse BMSCs, the cells were individually co-cultured with RAW264.7 cells and tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (three or more nuclei), and were subsequently counted under a microscope after 7 days. The results of five independent experiments are presented here.