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. 2017 Jul 5;7:4695. doi: 10.1038/s41598-017-04835-x

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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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Effect of pBMP-9 and SpBMP-9 on the metabolic enzymatic activity and indirect cell counting. (A) MTS assays of SH-SY5Y cells stimulated for 1d, 3d and 5d with equimolar concentrations (0.1 and 1 nM) of BMP-9, pBMP-9 and SpBMP-9 +/− 10 μM RA in serum-free culture medium (⬨p < 0.05 and ⬨⬨⬨p < 0.001 compared with the control without RA, *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control with RA). Results are the means ± SEM of at least two independent experiments performed in duplicate. (B) Indirect cell counting showing the relative fluorescence intensity of the stained nuclei in cells used to perform the MTS assays (*p < 0.05, ** p < 0.01, ***p < 0.001). (C) Indirect cell counting results plot in function of the MTS enzymatic assay results showing a correlation of 0.7. (D) Cell nucleus staining in SH-SY5Y cells stimulated by BMP-9, pBMP-9 or SpBMP-9 (0.1 and 1 nM) in the presence of 10 μM RA for 5d (Bar = 100 μm).