Fig. 3

Dependence of RPR-induced osteoclast differentiation on NF-κB stimulation. a Human monocytes, RAW264.7 macrophages, and K562 cells were developed with RPR proteins for 18 h. The MTT assay examined cell survival. Data are the mean ± SD of at least three separate tests;*p < 0.01; **p < 0.001, compared with the control for the agent alone. b RAW 264.7 cells were given RPR (10 μg/ml) or RANKL and M-CSF, with or without the NF-κB inhibitor (NF-κB SN50), pan-caspase inhibitor (Z-VAD-fmk), caspase-9 specific inhibitor (Z-LEHD-FMK), or caspase-3 specific inhibitor (Z-DEVD-FMK). After incubation for 7 days, a TRAP assay was utilized on the cells. Data are the mean ± SD of at least three independent experiments; **p < 0.001. c Murine RAW264.7 macrophages were transfected with an NF-κB luciferase reporter. After 24 h of transfection, cells were treated with RPR (100 ng/ml) or RANKL (100 ng/ml) for 14 h, then lysed for the luciferase assay. A dual-luciferase reporter assay system determined luciferase activity. Data are the mean ± SD of at least three samples. The results reflected at least three independent tests of the same experiment; *p < 0.01. d The effects of RPR (100 ng/ml) or RANKL (100 ng/ml) on nuclear NF-κB-p65 levels in murine RAW264.7 macrophages after 30 mins of treatment. RANKL activated NF-κB in RAW264.7. The agent alone did not affect NF-κB stimulation. Each band’s β-actin value normalized the densitometry reading