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. 2018 Oct 23;8:15647. doi: 10.1038/s41598-018-34092-5

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© The Author(s) 2018

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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Knockdown of Sema3A induces the formation of supernumerary axons. (a) HEK 293 T cells were transfected with vectors for FLAG-Sema3A (S3A) or RNAi-resistant FLAG-Sema3A-res (S3Ares) and an shRNA directed against Sema3A (S3A RNAi) or pcDNA6.2-GW/EmGFP-miR (Ctrl). The expression of Sema3A and GFP was analyzed by Western blot (WB) using an anti-FLAG antibody. The molecular weight is indicated in kDa. (b) Hippocampal neurons from E18 rat embryos were transfected with vectors for GFP (green), an shRNA against Sema3A (Sema3A RNAi) or pcDNA6.2-GW/EmGFP-miR (control), and a vector for RNAi-resistant FLAG-Sema3A-res (Sema3A-res) or pBK-CMV (control) as indicated. Neurons were analyzed at 3 d.i.v. by staining with an anti-MAP2 (red, dendrites) and the Tau-1 antibody (blue, axon). Representative images of transfected neurons are shown. The scale bar is 25 μm. (c) The percentage of unpolarized neurons without an axon (0, red), polarized neurons with a single axon (1, blue) and neurons with multiple axons (>1, green) is shown (Student’s t-test and two-way ANOVA; n = 3, independent experiments with >150 neurons per experiment; values are means ± s.e.m, ***p < 0.001 compared to control as indicated; n.s., not significant).