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. 2017 Dec 20;8:2226. doi: 10.1038/s41467-017-02368-5

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© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — The impaired adhesion structures in Ccr5-deficient (Ccr5 ^−/−) osteoclasts. a (left) A schematic illustration of the time schedule for mouse osteoclast culture from BMCs . (right) A schematic drawing shows the spatial locations of the actin ring (podosome belt) in a mature osteoclast cultured on a dentin slice. b Actin ring formation assays of wild-type (Ccr5 ^+/+) and Ccr5 ^−/− osteoclasts on dentin slices. Cells were stained with phalloidin-AlexaFluor568 (shown in red). The magnification of the upper and lower panels was ×20 (scale bars, 50 μm) and ×40 (scale bars, 10 μm), respectively (n = 5). The size and number of actin ring per area were scored and statistically compared. c Resorption pit assays. After matured, the osteoclasts were removed from the dentin slices and were subsequently stained with hematoxylin to visualize the resorption pits. Color images (upper panels) and gray-scale images (lower panels) are shown (scale bars, 100 μm). The resorption pit areas (%) on the gray-scale images were scored and statistically compared (n = 5). d The relative mRNA expression levels of Mmp3 and Mmp13 were measured by a real-time Q-PCR and statistically compared on day 5. The data shown as the mean ± SD (n = 5). e The formation of actin rings (podosome belts) in wild-type and Ccr5 ^−/− mature osteoclasts was visualized by enhanced resolution confocal imaging (left 4 images, scale bars, 200 μm). The cells were subjected to immunohistochemical staining with anti-tubulin antibodies (shown in red), and were concomitantly stained with phalloidin-AlexaFluor488 and DAPI to visualize the actin rings (shown in green) and nuclei (in blue), respectively (n = 4). SIM images (right two panels) demonstrate the assembly of actin-enriched podosome cores and vinculins in wild-type and Ccr5 ^−/− mature osteoclasts. The cells were subjected to immunohistochemical staining using anti-vinculin antibodies (shown in red), and were concomitantly stained with phalloidin-AlexaFluor488 to visualize the actin rings (shown in green, scale bars, 5 μm, n = 4). *P < 0.05 by Student’s t-test. All data are shown as the mean ± SD