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. 2017 Jun 2;7:2687. doi: 10.1038/s41598-017-02461-1

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© The Author(s) 2017

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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Cardiac endothelial cells proliferate in vitro. 24 h after seeding, ECs were cultured in EGM2 medium supplemented with 0, 0.5 or 10% FBS for 12 h and then analyzed for DNA synthesis using a 12 h pulse of EdU. (a) Representative images of endothelial cells stained for EGFP (green), EdU (red), and DAPI (nuclei, blue). White arrowheads point to EdU-positive endothelial cells. (b) Quantification of EdU+ EGFP+ endothelial cells without or with FBS in the culture medium (n = 3, mean ± SEM). P < 0.05 was considered statistically significant. (c) Example of cell density experiments. Cells were cultured with 10% FBS, resulting in endothelial cell proliferation. Plus and asterisk symbols are depicting landmarks, showing that pictures were taken from the same region for the cell density analysis. Yellow frames indicate areas of analysis. (d) Quantitative analysis of endothelial cell number increment in 24 h (n = 3, mean ± SEM). One way ANOVA followed by Bonferroni’s post-hoc test (GraphPad Prism) was performed to evaluate statistical significance of differences. P < 0.05 was considered statistically significant. ** corresponds to P < 0.05.