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. 2020 Feb 13;10:2513. doi: 10.1038/s41598-020-59363-y

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© The Author(s) 2020

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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Analysis of the effect of C-PC on RANKL-mediated osteoclast differentiation. (A) Effect of different doses of C-PC (10, 25, and 50 µg) on osteoclast differentiation. Phase-contrast microscopy images of TRAP stained osteoclasts are shown at indicated doses. Magnification is 4X. (B) The number of TRAP +ve multinucleated osteoclasts were counted in osteoclasts untreated (0) or treated with 10, 25, and 50µg C-PC for 72 h. Statistical analysis was performed to determine the dose-dependent inhibitory effect of C-PC on osteoclasts differentiation as compared with control untreated (0) cells. (C) Identification of the time-dependent effect of RANKL and C-PC on the osteoclast differentiation. The diagrammatic sketch demonstrates the treatment strategy of RAW cells with RANKL and C-PC (25 µg/ml). (D) Representative images of TRAP stained osteoclasts in response to the treatment strategy shown in panel C.TRAP stained osteoclasts in panels a,c,e, and g were taken with 4X objective (magnification: X40), while panels in b,d,f, and h were taken with 10X objective (magnification: X100). (E) The number of TRAP +ve multinucleated osteoclasts were counted in all treatment groups. Statistical analysis was performed to compare the number in the treatment groups to the control group. One-way ANOVA was applied, and values were expressed as mean ± SD. ***P < 0.001.