Vigeo affects receptor activator of nuclear factor-B ligand (RANKL)-induced phosphorylation of early signals and expression of c-FOS and NFATc1. (A) Bone marrow macrophages (BMMs)were seeded in the presence of M-CSF (10 ng/mL) for 1 day and then starved for 4 h. (A) Vigeo was used to pretreat the cells for 1 h followed by 0, 5, 15, and 30 min of RANKL stimulation. Whole-cell proteins were extracted by lysis using RIPA buffer and the protein expression was detected by western blotting using the indicated antibodies. (B) BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of Vigeo (100 μg/mL). Total RNA was isolated from the cells and mRNA expression levels of c-Fos and Nfatc1 were evaluated by real-time RT-PCR. ** p < 0.01, *** p < 0.001 versus the control; #
p < 0.05, ##
p < 0.01, ###
p < 0.001 versus the control at indicated times. (C) BMMs were cultured with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or absence of Vigeo (100 μg/mL). Protein expression was detected using western blotting with the indicated antibodies. β-actin was used as an internal control.