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. 2020 Mar 5;10(6):987–1003. doi: 10.1016/j.apsb.2020.02.015

Figure 3.

Figure 3

TIMP1 knockdown induces trans-endothelial barrier leakage and degradation of endothelial JPs. (A) Illustration of the in vitro BBB model to evaluate barrier function. An HBMECs monolayer seeded on top membrane of the cell culture insert followed by measurement of the concentration of 40 kDa FITC-dextran leaking into abluminal to determine the paracellular permeability (left) or the trans-endothelial electrical resistance (TEER) to estimate the TJ integrity (right). (B) HBMECs transfected with control or siRNA against hTIMP1 were subject to transwell permeability assay. Data represent mean ± SEM of six independent experiments (**P < 0.01). (C) HBMECs transfected with control or siRNA against hTIMP1 were subject to TEER assay. Data represent mean ± SEM of six independent experiments (**P < 0.01). (D) Western blot analysis and quantification of the indicated proteins in total cell lysates of HBMECs treated with siRNA. GAPDH was used as loading control. Data represent mean ± SEM of three independent experiments (ns, not significant; *P < 0.05; **P < 0.01). (E) Western blot analysis and quantification of the indicated proteins on membrane fragments of HBMECs treated with siRNA. Na/K ATPase was used as loading control. Data represent mean ± SEM of three independent experiments (**P < 0.01). (F) The distribution of junctional proteins in HBMECs treated with siRNA were analyzed by immunofluorescence (IF) staining using antibodies against VE-cadherin (green), claudin-5 (green), occludin (green) or claudin-5 (green) and counterstained with Hoechst33342 (blue) for nuclear labelling. Scale bars, 10 μm.