Figure 4.

The Effect of NFATc1 on Expression of Various Genes during Osteoclast Fusion Mediated by Other Known Fusion-Inducing Factors

A and B, To generate preosteoclasts, BMMs were cultured for 48 h with M-CSF and RANKL. Preosteoclasts were incubated for an additional 24 h with M-CSF, in the absence (Vehicle) or presence of RANKL, TNF-α, or LPS, with or without RANK-Fc (3 μg/ml) as indicated. A, Cultured cells were fixed and stained for TRAP. B, TRAP(+) MNCs having more than three nuclei were counted as osteoclasts. Data represent means ± sd of triplicate samples. C–E, Preosteoclasts were incubated for 24 h with M-CSF, in the absence (Vehicle) or presence of RANKL, TNF-α, or LPS, with or without CsA (1 μg/ml) as indicated. C, Cultured cells were fixed and stained for TRAP. TRAP(+) MNCs having more than three nuclei were counted as osteoclasts. Data represent means ± sd of triplicate samples. D, RT-PCR was performed to detect expression of the indicated genes. E, Cytoplasmic and nuclear fractions were harvested from cultured cells and subjected to SDS-PAGE and Western blot analysis for detection of NFATc1. Antibodies for actin and histone H3 were used for the normalization of cytoplasmic and nuclear extracts, respectively.