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. 2014 Jan 13;211(1):153–163. doi: 10.1084/jem.20130538

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© 2014 Hirose et al.

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Figure 3. — Stat5 suppresses the bone-resorbing activity of osteoclasts. (A) BMDMs from Stat5^fl/fl mice and Stat5 cKO mice were cultured in the presence of M-CSF and RANKL for 4 d, and cultures were stained for TRAP⁺ cells. (B) BMDMs from Stat5^fl/fl mice and Stat5 cKO mice were cultured in the presence of M-CSF and RANKL for 4 d, and the TRAP⁺ multinucleated cells (MNCs) containing more than three nuclei were counted as osteoclasts. Data are represented as the mean ± SD (n = 3). (C) Osteoclasts from Stat5^fl/fl and Stat5 cKO mice were cultured on dentine slices for 24 h, and the resorption areas were visualized by staining with toluidine blue staining and measured using an image analysis system. Representative resorption pits and TRAP staining of parallel cultures are shown (left). Graph shows pit resorption area per osteoclast (right). Data are represented as the mean ± SD (**, P < 0.01; n = 3). (D) Osteoclasts from Stat5^fl/fl or Stat5 cKO mice infected with AxGFP or AxStat5a/b were cultured on dentine slices for 24 h, and the resorption areas were visualized by staining with toluidine blue staining. Pit resorption area per osteoclast and representative resorption pits are shown. Data are represented as the mean ± SD (*, P < 0.05; n = 3). (E) Osteoclasts from WT mice infected with AxGFP or AxStat5a/b were cultured on dentine slices for 24 h, and the resorption areas were visualized by staining with toluidine blue staining. Graph shows pit resorption area per osteoclast. Data are represented as the mean ± SD (*, P < 0.05; n = 3). (F) TRAP staining and rhodamine-conjugated phalloidin staining of osteoclasts from Stat5^fl/fl mice and Stat5 cKO mice on dentin slices. (G) Stat5^fl/fl and Stat5 cKO osteoclasts were cultured for 8 or 24 h after cytokine withdrawal (left). Apoptosis of osteoclasts from Stat5^fl/fl and Stat5 cKO mice after 0- and 3-h serum starvation was assessed by TUNEL staining. Data are represented as the mean ± SD (n = 3). (A, C, D, F, and G) Bars, 100 µm. Results in all panels are representative of two independent experiments.