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. 2021 Apr 21;11:8585. doi: 10.1038/s41598-021-87949-7

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© The Author(s) 2021

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Netrin-1 inhibits adipogenesis and chondrogenesis in vitro. (a–e) Netrin-1 inhibits MEF adipogenesis. Wild-type MEFs were induced with an adipogenic cocktail together with or without 20 ng/ml BMP4 and/or 0.3 µg/ml netrin-1. After nine days, the cells were fixed, stained with Oil Red O, and imaged, and the staining was quantified. (a–d) Representative micrographs of MEFs treated with the adipogenic cocktail together with (a) no further additions, (b) BMP4, (c) BMP4 and netrin-1, and (d) netrin-1. Scale bars, 0.6 mm. (e) Quantification of the Oil Red O-stained areas as outlined in a-d. Shown are the means and individual values of eight independent experiments. (Student’s t-test: ***p < 0.001). (f) Netrin-1 inhibits chondrogenesis. ATDC5 cells were stimulated with BMP4 alone or in combination with different concentrations of netrin-1 for 72 h. Thereafter, chondrogenesis was assessed via an alkaline phosphatase (ALP) assay. The graph represents the average means with standard deviations of four independent experiments performed in duplicate. (One-way ANOVA: ^####p < 0.0001. Post-hoc test with multiple comparisons [compared to netrin-1, 0 ng/ml] adjusted p-value: **p < 0.01; ***p < 0.001; ****p < 0.0001).