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. 2019 Feb 28;9:3099. doi: 10.1038/s41598-019-39430-9

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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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Claudin-4 knockdown reduces TEER and increases absolute permeability to both sodium and chloride. (A) mIMCD3 cells expressing kAE1, Cldn-4/EV or Cldn-4/kAE1 in an inducible manner were either incubated in presence or absence of doxycycline for 48 hours, prior to lysis and analysis of protein abundance by immunoblot. Mouse anti-HA antibody was used to detect kAE1 protein (upper panel), rabbit anti-claudin-4 (middle panel) or rabbit anti-claudin-8 (bottom panel) antibodies were used respectively to detect claudin-4 and claudin-8. The dark circle corresponds to kAE1 carrying complex oligosaccharide, and the white circle indicates kAE1 carrying high mannose oligosaccharide. Twenty micrograms of proteins were loaded in every lane. (B) Quantitative PCR analysis of claudin-4 mRNA abundance in Cldn-4 KD/kAE1 incubated in control conditions or with Doxycycline. The results are expressed as a percentage of “Cldn-4 KD/kAE1 – Dox” conditions and are normalized to the expression of actin. Error bars correspond to means ± SEM, n = 3. *P < 0.05 versus “Cldn-4 KD/kAE1 - Dox” condition using un-paired t-test. (C) Ussing chambers data showing an equivalent decrease in TEER in both Cldn-4 KD/EV and Cldn-4/kAE1 cells incubated with doxycycline. Error bars correspond to means ± SEM, n = 4. ***P < 0.001 versus Cldn-4 KD/kAE1 cells without doxycycline, ****P < 0.0001 versus Cldn-4 KD/EV cells without doxycycline. n.s. indicates non significant difference (One-way ANOVA). Although P_Na / P_Cl ratio was unchanged, (D), absolute permeability to both sodium (P_Na) (E) or chloride (P_Cl) (F) were increased upon Doxycycline incubation of Cldn-4/EV and Cldn-4/kAE1 cells. Error bars correspond to means ± SEM, n = 4, ***P < 0.001 versus Cldn-4 KD/EV cells without doxycycline, **P < 0.01 versus the same cell line without doxycycline.