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. 2022 Jul 8;13:3960. doi: 10.1038/s41467-022-31697-3

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© The Author(s) 2022

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. 5 — a, b Immunofluorescence images of Sox9 and Col2a1 in the growth plate of Mast4^+/+ and Mast4^−/− mice at PN 1 day (n = 3). c, d Pentachrome staining of the tibial growth plates of the (c) 1-day-old and (d) 3-week-old Mast4^+/+ and Mast4^−/− mice (n = 5). Black arrows indicate the hypertrophic zone. e The μCT images of the trabecular bone in the tibias of Mast4^+/+ and Mast4^−/− mice. Total bone volume and bone mineral density (BMD) of trabecular bone were measured from the μCT images (n = 8 for Mast4^+/+ and 10 for Mast4^−/− mice). f Bone formation was visualized by double calcein labelling at 7d and 2d prior to sacrifice, and the distance between labels were measured at 3 points per tibia (n = 10 tibias for Mast4^+/+ and 5 tibias Mast4^−/− mice). MAR: mineral apposition rate. g Representative images of the immunofluorescence staining of Osterix and Mmp13 near metaphyseal blood vessels (CD31) in the proximal tibia of 6-week-old Mast4^+/+ and Mast4^−/− mice (n = 3). GP: growth plate, MP: metaphysis. h Representative alcian blue and Alizarin Red S staining results obtained from in vitro colony formation assay using skeletal stem cells isolated from 5-week-old Mast4^+/+ and Mast4^−/− mice (n = 5). e, f, Data are reported as mean ± SD. Unpaired two-tailed Student’s t test (P < 0.05) was conducted for all statistical analyses. Source data for e, f are provided as a Source Data file.