Fig. 2

TCG suppressed RANKL and M-CSF induced osteoclast differentiation in vitro. A, B BMMs were induced into osteoclasts with RANKL and M-CSF while simultaneously treated with indicated doses of TCG. A Osteoclasts were visualized using TRAP staining (Scale bar = 500 µm). B Cells with more than 3 nuclei were considered as osteoclasts and included in statistics. C BMMs were treated with indicated doses of TCG for a specified period of time, the cytotoxicity of TCG on BMMs was assessed using CCK8 assay. D–K BMMs were induced with M-CSF and RANKL, and simultaneously treated with or without 1 µM TCG for a specified period of time. D, E Western blot measured the protein expression of TRAF6, NFATc1, c-Fos and the phosphorylation of MAPK and NFκB signaling molecules, the relative protein expression statistics were presented in Additional file 1: Fig. S1; F–K RT-PCR was conducted to assess the transcriptional levels of key genes involved in osteoclast differentiation. L, M Osteoclasts were seeded onto cortical bone slices and treated with indicated dose of TCG for 48 h. L Bone resorption pits appear cloud-like under confocal microscopy, as indicated by red arrows (Scale bar = 100 µm). M The area of bone resorption pits was quantified using ImageJ, and the percentage of bone resorption area was calculated by dividing the pit area by the total bone slice area. Data were presented as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.01