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. 2020 Aug 26;17:53. doi: 10.1186/s12987-020-00212-5

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© The Author(s) 2020

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/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 2 — Comparison of cell morphology and localization and expression of claudin-5 and Pgp in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCECs. a Claudin-5 was indirectly stained (hCMEC/D3-WT and pBCECs) or visualized by YFP tag in the stably transduced hCMEC/D3-Cldn5-YFP cell line (for better visualization both claudin-5 and Cldn5-YFP are depicted in green). F-Actin is shown in red and cell nuclei are counterstained in blue by DAPI. b An xz scan of the cell layer revealed interendothelial (junctional) localization of claudin-5 (green) in the two cell lines and pBCECs. Scale bars: 10 µm. c Phase contrast micrographs of hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and primary pBCEC cultures. d Purity of pBCEC isolation, evaluated by fluorescent staining for the endothelial cell marker CD31 (green). Cell nuclei were counterstained with DAPI (blue). e Length and width of hCMEC/D3 and pBCECs. f Representative Western blot showing claudin-5 (Cldn5) expression in hCMEC/D3-WT (7 days after seeding), hCMEC/D3-Cldn5-YFP (7 and 21 days after seeding) and pBCEC cultures (7 days after seeding). β-actin was used as a loading control. g Quantification of Western blot bands and normalization of claudin-5 expression to β-actin. Data are represented as mean ± SEM of n = 3 independent experiments. Significant intergroup differences are indicated by asterisk (*P < 0.05). h Representative Western blot showing Pgp expression in hCMEC/D3-WT, hCMEC/D3-Cldn5-YFP and pBCEC cultures. β-actin was used as a loading control. i Quantification of Western blot bands and normalization of Pgp expression to β-actin. Data are represented as mean ± SEM of n = 3 independent experiments. Significant intergroup differences are indicated by asterisk (*P < 0.05)