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. 2020 Aug 19;28(1):283–302. doi: 10.1038/s41418-020-0600-6

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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. 2 — a Transfection efficiency was evaluated by qPCR and showed upregulation of circRNA-vgll3 expression in the Lenti-circRNA-vgll3-transfected group. The transfection efficiency was not influenced by RNase R treatment (n = 3, *P < 0.05 versus Control group, statistical analysis was performed by Student’s t test). b Three shRNAs for circRNA-vgll3 were cloned into vectors, and qPCR analysis showed that circRNA-vgll3 inhibitor 1 and 3 can effectively knockdown the expression of circRNA-vgll3. The knockdown efficiency was not influenced by RNase R treatment (n = 3, *P < 0.05 versus Control group, statistical analysis was performed by one-way ANOVA). c–d mRNA expression levels of the markers Runx2, OSX, Col1a1, OPN, OCN, and BSP were elevated at osteogenesis day 7 by Lenti-circRNA-vgll3 whereas they were impaired by circRNA-vgll3 inhibitor when compared to Lenti-NC (n = 3, *P < 0.05 versus Control group, statistical analysis was performed by Student’s t test). e Western blot analysis detecting the levels of Runx2, Col1a1, BSP and OCN at osteogenesis day 7 showed that they were significantly promoted by Lenti-circRNA-vgll3 and greatly attenuated by transfection of the circRNA-vgll3 inhibitor in comparison to Lenti-NC. f ALP staining evaluated the effect of circRNA-vgll3 on ALP activity at osteogenesis day 7. Scale bars: 100 µm. g ARS staining evaluated the effect of circRNA-vgll3 on the ECM mineralization at osteogenesis day 14. Scale bars: 100 µm. h–i Semiquantitative analysis of ALP activity and ARS activity (n = 3, *P < 0.05 versus Control group, statistical analysis was performed by one-way ANOVA). j Cellular immunofluorescence showing the cellular expression levels of Runx2, OPN and BSP at osteogenesis day 7 (n = 3, *P < 0.05 versus Control group, statistical analysis was performed by one-way ANOVA, Scale bars: 100 µm).