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. 2021 Aug 30;156(6):609–616. doi: 10.1007/s00418-021-02026-4

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© The Author(s) 2021

Open AccessThis 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. 1 — Differentiation of junctional properties of Caco2 cells. a Representative immunostainings during differentiation of the epithelial barrier of clone 1 are shown. At confluence, in contrast to E-cadherin, junctional proteins Desmoglein-2 (Dsg2) and Claudin-1 were not regularly located at the cell borders. This changed during differentiation of the monolayer to the state of full barrier 14 days later when all junctional proteins showed a regular staining at the cell borders (representatives are shown for n = 6, scale 20 µm). b Similar observations for the junctional proteins were seen for clone 2, where tight junction protein Claudin-1 was hardly detectable at the state of confluence but showed a linear staining at the cell borders at the moment of barrier differentiation after 4 days (representatives are shown for n = 6, scale 20 µm). c. Permeability of 4 kDa FITC-dextran revealed that, with the differentiation of junctional proteins of clone 1, the permeability coefficient (P_E) declined to (0.31 ± 0.11) × 10^–6 cm/s from (3.32 ± 0.13) × 10^–6 cm/s at the state of confluence. This was not further altered under full barrier conditions 21 days after confluence where P_E was (0.36 ± 0.10) × 10^–6 cm/s (n = 6, *p < 0.05, one-way ANOVA). d. The measurements of P_E for clone 2 paralleled the development of clone 1. At confluence, P_E was (1.64 ± 0.26) × 10^–6 cm/s and dropped to (0.35 ± 0.19) × 10^–6 cm/s 4 days post-confluence, which was not changed at full barrier properties after 6 days (n = 6, *p < 0.05, one-way ANOVA). e. Transepithelial electric resistance (TEER) of clone 1 during the differentiation of the cell monolayer is shown. After seeding, TEER was steadily increasing to 137 ± 16 Ω cm². Then, over the next days, TEER decreased to 88 ± 7 Ω cm² before it increased again to 139 ± 18 Ω cm² until the state of full barrier was reached (n = 10, *p < 0.05, two-way ANOVA). f. This change of TEER over the time was almost identical in clone 2, where TEER rose to 87 ± 2 Ω cm² at confluence, dropped during differentiation of the Caco2 cells, and then increased to 91 ± 1 Ω cm² at full barrier (n = 10, *p < 0.05, two-way ANOVA)