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. 2013 Sep 23;210(10):1947–1960. doi: 10.1084/jem.20130512

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© 2013 Maruyama et al.

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Figure 5. — Impaired multinucleation of Sbno2^−/− osteoclasts. (A) MDMs from wild-type and Sbno2^−/− mice were cultured in the presence of the indicated RANKL concentrations. Representative TRAP staining is shown. Bar, 200 µm. (B) MDMs from wild-type and Sbno2^−/− mice were cultured in the presence of the indicated RANKL concentrations. After 4 d, TRAP⁺ MNCs (more than three nuclei) were counted. (C) MDMs from wild-type and Sbno2^−/− mice were cultured in the presence of 50 ng/ml RANKL, and sequential TRAP staining was performed to count the numbers of osteoclasts. (D) Apoptosis of wild-type and Sbno2^−/− MDMs. Caspases were stained using a Poly Caspase Assay kit (ImmunoChemistry Technologies), and the numbers of caspase-positive cells were counted under a fluorescence microscope. (E) Analysis of wild-type and Sbno2^−/− MDMs using the F4/80 and CD11b markers. (F) Resorption pits (analyzed by scanning electron microscope, arrowheads) from wild-type and Sbno2^−/− osteoclasts. MDMs were plated on dentine slice and cultured with 50 ng/ml RANKL for 5 d. Pit numbers and multinucleated cell numbers were counted (middle). Resorption area (right) was determined using Image J. Bar, 100 μµm. (G) F-actin was stained with phalloidin. Bars: (left) 100 µm; (right) 5 µm. (H) MDMs from wild-type and Sbno2^−/− mice were cultured in the presence of 25, 50, or 75 ng/ml RANKL. After 4 d, the number of nuclei in TRAP⁺ cells was analyzed. (I) Bone marrow cells (OC) and calvariae (OB) were cocultured in the presence of 1α,25(OH)2D3 for 8 d. TRAP⁺ MNCs were counted. (J) MDMs from wild-type and Sbno2^−/− mice were cultured in the presence of 100 ng/ml IL-4. After 4 d, TRAP⁺ MNCs (more than three nuclei) were counted. Arrowheads indicate MNCs. (K) In vivo foreign body formation was induced by subcutaneous insertion of coverslips. After 4 d, coverslips were harvested and stained by hematoxylin and eosin. MNCs/ coverslips (n = 6) and numbers of nuclei/MNC (wild-type: n = 45, Sbno2^−/−: n = 31) were quantified. Arrowheads indicate MNCs. Results are representative of four (A–C) or three (D–J) independent experiments. Error bars, SE. *, P < 0.05, n = 4, unless indicated.