The inhibitory effect of NDGA on osteoclast differentiation in vitro. (a) Dose dependency of NDGA on osteoclastogenesis in RAW-D cell culture (RAW-D). RAW-D cells were cultured for 3 days for forming osteoclast-like MNCs in the presence or absence of the indicated concentrations of NDGA. Upper panel, demonstration of cultures. Original magnification × 75. (b) Dose dependency of NDGA on osteoclastogenesis in BMM culture. Mouse BMMs (mBMMs) were cultured for 5 days for forming osteoclast-like MNCs. Indicated concentrations of NDGA were added to the culture. (c) MTT assay. The left panel: RAW-D cells or mBMMs were cultured for forming osteoclasts, as described above, in the presence or absence of the indicated concentrations of NDGA (+RANKL). MTT assay was performed at 3 days in RAW-D cells and at 5 days in mBMMs, respectively. The right panels: MTT assay was performed also in the absence of RANKL (−RANKL). (d) Suppression of osteoclastogenesis by NDGA in rat bone marrow (rBM) culture system. The rBM cells were cultured for forming osteoclasts as described in Materials and Methods for 4 days followed by staining for TRAP and by osteoclast-specific mAb Kat1. In a, b, and d, after staining for TRAP, number of TRAP-positive MNCs was counted. In d, number of Kat1-positive MNCs was also counted. Data represent means±s.d. (n=4). Data were analyzed by Student’ s t-test. **P<0.01 and ***P<0.001 compared with cultures in the absence of NDGA. (e) Effect of NDGA on gene expression associated with osteoclastogenesis. RAW-D cells were cultured for forming osteoclasts, as described above, in the presence or absence of the indicated concentrations of NDGA. Total RNA was extracted and the mRNA expression levels of the indicated genes were estimated by RT-PCR.