Figure 1.

The effect of ciclopirox on receptor activator of nuclear factor kappa B ligand (RANKL)-mediated osteoclastogenesis. (A) Primary bone marrow macrophages (BMMs) were cultured in the presence of macrophage colony-stimulating factor (M-CSF) (10 ng/mL), RANKL (20 ng/mL), and various concentrations of ciclopirox for 4 days. The cells were stained for Acid Phosphatase, Leukocyte (TRAP). (B) The number of TRAP-positive multinucleated cells (≥3 nuclei) was calculated. (C) BMMs were incubated with M-CSF (10 ng/mL) and various concentrations of ciclopirox for 3 days. The methylthiazolyldiphenyl-tetrazolium bromide assay was performed to evaluate cell viability. (D–G) BMMs were cultured with M-CSF (10 ng/mL) and RANKL (20 ng/mL) in the presence or absence of 2.5 μM ciclopirox for 4 days. (D) The cells were fixed and probed with anti-nuclear factor of activated T cells cytoplasmic 1 (NFATc1) antibody. The nuclei and F-actin were then labeled with DAPI and rhodamine-conjugated phalloidin, respectively. Scale bar, 100 μm. Quantification of the percentage of (E) cells displaying actin rings and (F) cells expressing nuclear NFATc1. (G) The relative expression levels of osteoclast marker genes were measured by quantitative RT-PCR. Each experiment was performed three times independently. ** p < 0.01 (two-tailed Student’s t-test).