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. 2019 Mar 21;9:4982. doi: 10.1038/s41598-019-41442-4

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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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Human and experimental NEC is associated with changes in claudin 2 localization within the cell. (a) Caco-2 cells treated with LPS for 7 days were fractionated into subcellular compartments and analyzed with western blot showing a decrease in claudin 2 expression in the membrane of LPS-treated cells vs. controls (n = 5/4) and increased expression in the cytoskeleton vs. controls (n = 3/4); representative immunoblots for claudin 2 per compartment shown (full blots included in Supplementary Fig. S3). (b) Representative immunofluorescence micrographs of human and rat intestinal villi stained for claudin 2 (red) show focal expression at the cell membrane in control groups vs. internalized pattern of expression in NEC groups; nuclei stained with DAPI (blue), scale bar = 20 uM. (c) Mean fluorescent intensity measured in 15 × 30 um size boxes (2-cells wide) showed increased claudin 2 fluorescence in humans and rats NEC compared to controls (n = 3/3). All values are mean ± SEM (*p < 0.05, **p < 0.01, ***p < 0.001).