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. 2019 Aug 19;9:12009. doi: 10.1038/s41598-019-48519-0

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© The Author(s) 2019

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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Ephrin-A5 increases Neogenin and EphA4 protein levels in LMC growth cones. (a) Mean Neogenin IF in LMC explants treated 15′ with either a control solution containing Fc at 500 ng/mL or ephrin-A5 at concentrations ranging from 10 to 500 ng/mL. Ephrin-A5 at 50 and 100 ng/mL results in increased levels of Neogenin IF (ephrin-A5 50 ng/mL: 1.7 ± 0.3-fold, p = 0.038; ephrin-A5 100 ng/mL: 2 ± 0.3-fold, p = 0.011). (b–e) Examples of Neogenin IF in growth cones quantified in (a). (f) Mean Neogenin IF in growth cones of LMC explants treated with either Fc or ephrin-A5 at 100 ng/mL for 10′ 20′ and 40′. A 10′ exposure to ephrin-A5 is sufficient to increase Neogenin IF (1.4 ± 0.1-fold, p = 0.0007) and the increase is maintained after 20′and 40′ (1.3 ± 0.1-fold, p = 0.0017; 1.3 ± 0.1-fold, p = 0.0095 respectively). (g) A 5′ exposure to ephrin-A5 at 100 ng/mL is sufficient for increasing the mean Neogenin IF in LMC growth cones (1.7 ± 0.2-fold, p = 0.0228). (h) Quantification of mean Neogenin, EphA4, Unc5c and BEN IF levels in growth cones exposed to either MN media or ephrin-A5 at 100 ng/mL for 20′. Ephrin-A5 induced an increase in Neogenin and EphA4 IF (1.6 ± 0.1-fold, p < 0.0001; 2 ± 0.2-fold, p = 0.0001 respectively), Unc5c and BEN IF levels did not significantly differ (p = 0.0806 and p = 0.1391 respectively). (i,p) Examples of growth cones quantified in (h). Data are shown as mean ± SEM, statistical significance was tested using a two-tailed unpaired sample t-test. (a) Fc 500 ng/mL N = 5, ephrin-A5 10 ng/mL N = 3, eprhrin-A5 50 ng/mL N = 5, ephrin-A5 100 ng/mL N = 5, ephrin-A5 250 ng/mL N = 5, ephrin-A5 500 ng/mL N = 3; (f) Fc 100 ng/mL 40′ N = 4,ephrin-A5 100 ng/mL 10′ N = 4, ephrin-A5 100 ng/mL 20′ N = 4, ephrin-A5 100 ng/mL 40′ N = 4; (g) Fc 500 ng/mL 5′ N = 4, ephrin-A5 500 ng/mL 5′ N = 4; n: EphA4 N = 16, Neogenin N = 13, BEN N = 5, Unc5c N = 10. Values for the total number of growth cones and SEM values for each treatment for this figure and subsequent figures are provided in the Supplementary Excel File.