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. 2018 Feb 27;8:3719. doi: 10.1038/s41598-018-22017-1

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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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Constitutive expression of EGFP-EspF and EGFP-Map in MDCK cells disrupts the tight junction barrier. (A) Untransfected cells or cells constitutively expressing EGFP vector alone, EGFP-Tir, EGFP-EspF and EGFP-Map were cultured on polycarbonate Transwell filters and TER was measured daily to monitor the integrity of tight junctions. The maximum values of TER for each cell line are indicated. The paracellular flux of 4 kDa dextran (B) and 70 kDa dextran (C) was measured and plotted as fold change with respect to untransfected MDCK cells (normalized to 1). Error bars represent means ± s.e.m from three independent experiments; *indicates p-value < 0.01 and **indicates p-value < 0.001. (D) The cellular localization of TJ proteins was examined using the indicated primary antibodies and Cy3-conjugated secondary antibodies. Nucleus was labeled with DAPI (4′,6-Diamidino-2-phenylindole dihydrochloride). Constitutive expression of EGFP-EspF and EGFP-Map caused the reorganization of claudin-1, -4, occludin and ZO-1. Scale: 10 µm; UT: Untransfected.