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. 2018 Jun 1;9:2143. doi: 10.1038/s41467-018-04464-6

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© The Author(s) 2018

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. 6 — P2rX7 activation rescues impaired Tet DKO BMMSCs and osteopenia phenotype in Tet DKO mice. a The levels of miR-297a-5p, miR-297b-5p, and miR-297c-5p in control, Tet DKO BMMSCs, and P2rX7 CRISPR activation plasmid-treated Tet DKO BMMSCs. b Bradford assay showed the exosome secretion in control, Tet DKO BMMSCs, and P2rX7 CRISPR activation plasmid-treated Tet DKO BMMSCs. c Western blotting showed the expression of CD9 and CD81 in control, Tet DKO BMMSCs, and P2rX7 CRISPR activation plasmid-treated Tet DKO BMMSCs. d, e Mineralized nodule formation and the expression of Runx2, ALP, and OCN in control, Tet DKO BMMSCs, and P2rX7 CRISPR activation plasmid-treated Tet DKO BMMSCs, as assessed by alizarin red staining (d) and western blotting (e). f The expression of miR-297a-5p, miR-297b-5p, and miR-297c-5p in control, Tet DKO BMMSCs, and adeno-associated P2rX7 overexpression virus (P2rX7 AAV)-treated Tet DKO BMMSCs, as assessed by qPCR. g Bradford assay showed the exosome secretion level in control, Tet DKO BMMSCs, and P2rX7 AAV-treated BMMSCs. h Western blotting showed the expression of CD9 and CD81 in exosomes derived from control, Tet DKO BMMSCs, and P2rX7 AAV-treated BMMSCs. i Bone mineral density (BMD) of trabecular bone (TB) area in distal femurs of control, Tet DKO, and P2rX7 AAV-treated-mice, as assessed by micro-CT. j The cortical bone area (Ct.Ar) and cortical thickness (Ct.Th) of the femurs of control, Tet DKO, and P2rX7 AAV-treated-mice analyzed by micro-CT. k, l H&E staining showed the TB volume (yellow-circled area) of control, Tet DKO, and P2rX7 AAV-treated-mice. m The capacity of BMMSCs isolated from control, Tet DKO, and P2rX7 AAV-treated-mice to form mineralized nodules as assessed by alizarin red staining. n Western blotting analysis showed the expression levels of the osteogenic markers Runx2, ALP, and OCN in control, Tet DKO, and P2rX7 AAV-treated Tet DKO BMMSCs. The 8–10-week-old Tet1^−/−Prx1^creTet2^fl/fl mice were used as Tet DKO mice in our experiments, and their littermates whose genetic status was Prx1^cre were used as controls. *p < 0.05, **p < 0.01, ***p < 0.001 (mean ± SD). Scale bars, 400 μm (i, j), 1 mm (k). Results are from three independent experiments. p values were calculated using one-way ANOVA