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. 2023 Oct 5;14:6214. doi: 10.1038/s41467-023-41999-9

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© The Author(s) 2023

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Immunoblotting for CLDN23 and Calnexin (loading control) in SKCO15 cells with ectopic expression of full-length human CLDN23 protein versus a 10 amino acid myc-tag protein (Control). b Representative graph showing TEER of SKCO15 cells overexpressing CLDN23 vs control monolayers was measured continuously for 5 days. Data are mean ± SD and represent three individual experiments, each with six technical replicates. ****p ≤ 0.0001; two-way ANOVA with Tukey’s posttest. c Left, paracellular flux rate of 4 kDa TRITC-dextran (TD4) and 70 kDa FITC-dextran (FD70) across monolayers overexpressing CLDN23 and control SKCO15 cells ([Dextran]_basal). Right, rate of change of TD4 flux was utilized to calculate the apparent permeability (P_app) of each individual sample. Data are mean ± SD and are representative of three individual experiments, each with six technical replicates. ***p = 0.001; two-tailed Student’s t test with Welch’s correction. d CLDN23 expression in T84 IECs transduced with two shRNAs against CLDN23 were compared with scramble non-silencing shRNA control cells (NS). Immunoblot images are representative of three independent experiments. e Representative graph showing TEER of T84 CLDN23 KD (shRNA1 and 2) and NS monolayers was measured continuously for 5 days. Data are mean ± SD and represent two independent experiments, each with four technical replicates per condition. ***p ≤ 0.001; ****p ≤ 0.0001; two-way ANOVA with Tukey’s posttest. f Paracellular flux rate of 4 kDa TRITC-dextran (TD4) and 70 kDa FITC-dextran (FD70) across monolayers of T84 CLDN23 KD (shRNA1 and 2) and NS control monolayers ([Dextran]_basal). Rate of change of TD4 flux was utilized to calculate the apparent permeability (P_app) of each individual sample. Data are mean ± SD and represent two individual experiments. Each point represents an individual cell monolayer (n = 8 (NS), 6 (shRNA1), and 8 (shRNA2)). **p = 0.0068 (NS vs shRNA1), **p = 0.0015 (NS vs shRNA2); two-tailed Student’s t test. g Left, Cldn23 mRNA expression in Cldn23^ERΔIEC or Cldn23^f/f IECs. Points represent values from individual mice. Data are mean ± SD and represent two independent experiments, 4 mice per group. ****p ≤ 0001; two-tailed Student’s t test. Right, immunoblotting for CLDN23 and Calnexin (loading control) in colonic IECs from Cldn23^ERΔIEC and Cldn23^f/f mice. h Confocal images of colonic tissue sections of Cldn23^ERΔIEC and Cldn23^f/f mice stained for CLDN23 (green) and nuclei (DAPI/blue). Scale bar: 50μm. i Left, schematic of the intestinal loop model used to assess intestinal epithelial permeability to 4 kDa FITC dextran in vivo in Cldn23^ERΔIEC and Cldn23^f/f mice. Right, CLDN23 depletion resulted in increased intestinal permeability to 4 kDa FITC dextran in vivo. Histograms represent the mean ± SD from three independent experiments. Each point represents an individual mouse (n = 14 (Cldn23^f/f) and 16 (Cldn23^ERΔIEC)). ****p < 0.0001; two-tailed Student’s t test with Welch’s correction.