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. 2018 Mar 21;8:4986. doi: 10.1038/s41598-018-22940-3

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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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E-cadherin is essential for tether formation. 4T1 cells, stably knocked down for E-cadherin (shEcad), and control cells (ShCon) were imaged by DIC (A,D) and double-labelled for E-cadherin (B,E) and β-catenin (C,F). Note that tether formation and E-cadherin-rich adhesions are markedly reduced in the knocked-down cells. (G) QRT–PCR analysis, pointing to an over 85% reduction in E-cadherin mRNA levels in shEcad compared to shCon 4T1cells, obtained in 3 independent experiments. (H) Western blotting revealed an ~83% decrease of E-cadherin protein levels in 4T1-shEcad, compared to 4T1-shCon. (I) Comparison of the percentage of tether-associated cells in shEcad and shCon 4T1cells, pointing to an ~68% reduction in the knocked-down cells, compared to controls. ***p < 0.001. Scale bar: 10 μm. n = 11 different images (containing around 800–900 cells in total for each group), obtained in two independent experiments.