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. 2019 Feb 14;9:2030. doi: 10.1038/s41598-018-38322-8

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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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Effects of TNF-α and adalimumab on paracellular permeability, OCCLUDIN and ZO-1 in 3D Caco-2 cysts. Caco-2 cysts were exposed at the basal side to medium only (Ctrl), TNF-α (25 pg/mL) with or without pre-incubation of adalimumab (20 μg/mL) for 24 hours. (a) Representative images showing the intraluminal accumulation of FITC-dextran-4 (FITC-D4, green) in the Caco-2 cysts. Bar indicates 50 μm. (b) The mean FITC-D4 intensity of Caco-2 cysts measured and expressed as the L/BL ratio of the luminal (L) over the basal (BL) compartment. (c) mRNA levels of OCCLUDIN and ZO-1 in 3D Caco-2 cysts. (d–f) Confocal microscopy analyses of ZO-1 (green), OCCLUDIN (red) and nuclei (blue). Images in (d) were captured from the middle cross-section of 3D cysts. Bar indicates 50 μm. Images in (e) were captured from the top surface of 3D cysts. Bar indicates 20 μm. Quantified fluorescence intensities in (d) are shown in (f). All graphs represent the results of three replicate experiments. Data expressed as means ± SEM with at least 8 cysts per treatment. *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey’s post-hoc test.