MHR disrupts BEC integrity on the level of tight junction proteins.

<p>BEC displayed a physiological arrangement of TJ proteins Claudin 5 (<b>A</b>) and zonula occludens 1 (<b>B</b>) in a co-localizing manner (<b>C</b>) with only few TJ gaps (arrowheads). MHR disrupted the continuous arrangement of both junctional proteins in BEC (arrowheads <b>D-F</b>). Here, DPI blocked TJ injury in MHR, only few TJ disruptions were detected in BEC pre-treated with the NADPH oxidase inhibitor DPI (arrowheads <b>G-I</b>). Representative images from 3 experiments per group are shown. Quantitative analyses of Cl5 (Cl5 normoxia: 5.85 <u>+</u> 0.33 vs. Cl5 MHR: 2.72 <u>+</u> 0.54, P < 0.0001; n = 12 cells per group from 3 experiments, <b>J</b>) and ZO-1 (ZO-1 normoxia: 12.53 <u>+</u> 1.27 vs. ZO-1 MHR: 3.98 <u>+</u> 0.67, P < 0.0001; n = 12 cells per group from 3 experiments, <b>K</b>) integrity on cell membranes revealed that both junctional proteins are significantly perturbed in MHR. DPI pre-treatment significantly restored TJ integrity at cellular membranes under MHR (Cl5 MHR: 2.72 <u>+</u> 0.54 vs. Cl5 MHR+DPI: 6.57 <u>+</u> 0.69, P = 0.0002; ZO-1 MHR: 3.98 <u>+</u> 0.67 vs. ZO-1 MHR+DPI 10.55 <u>+</u> 1.21, P < 0.0001; n = 12 cells per group from 3 experiments).