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. 2020 Jan 2;8:2. doi: 10.1038/s41413-019-0073-8

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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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Fig. 5 — a, b Colony forming unit-fibroblast (CFU-F) assays, followed by Giemsa staining (a). The number of CFU-F was counted under a microscope (b). *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± standard deviation (s.d.). N = 3 mice per group. (c, d) Colony forming unit-osteoblast (CFU-OB) assays. *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± standard deviation (s.d.). e–g In vitro osteoblastic or adipogenic differentiation of primary bone marrow stromal cells (BMSCs), followed by Oil Red O staining (g). *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± s.d. Scale bar, 80 mm. h Real-time RT-PCR (qPCR) analyses. *P < 0.05, versus controls, Student’s t-test. N = 4 mice per genotype. The results are expressed as the mean ± s.d. i Real-time RT-PCR (qPCR) analyses. *P < 0.05, versus controls, Student’s t-test. N = 4 mice per genotype. The results are expressed as the mean ± s.d. j, k Immunohistochemical (IHC) staining of tibial diaphyseal cross-sections of 2-month-old male Kind2-D1 and their control littermates with an antibody against sclerostin. Scale bar, 40 mm. N = 8 control mice; N = 7 Kind2-D1 mice. *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± s.d. l, m Immunohistochemical (IHC) staining of tibial metaphyseal longitudinal sections of 2-month-old male Kind2-D1 and control littermates with an antibody against active b-catenin. Scale bar, 40 mm. N = 8 control mice; N = 7 Kind2-D1 mice. *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± s.d. n Western blot analysis. o Real-time RT-PCR (qPCR) analyses. N = 3 control mice; N = 4 Kind2-D1 mice. *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± s.d. p qPCR analysis. *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± s.d. q Crispr/Cas-9 deletion of Kindlin-2 in MLO-Y4 osteocyte-like cells. r qPCR analysis. *P < 0.05, versus controls, Student’s t-test. The results are expressed as the mean ± s.d. (s–u) MC-4 preosteoblastic cells were cultured in osteoblast differentiation media in the presence of the conditioned media from parent MLO-Y4 cells (WT-CM) or Kindlin-2-deficient subclone #10 (KO-CM) for 5 d, followed by western blotting with the indicated antibodies (s) or qPCR analysis of the indicated genes normalized to Gapdh mRNA (t), or 10 d, followed by Alizarin red staining (u).