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Fig. EV4 Characterization of the zebrafish embryo model. (A) OCLN-expressing RNA for oclna and oclnb paralogues assessed in the endothelium of Tg(fli:egfp) zebrafish embryos from (Bonkhofer et al, 2019). Kdr, kdrl, gapdh, and actb2 genes were used as housekeeping genes. (B) Comparative analysis of the human OCLN (hOCLN) and danio rerio (zebrafish) OCLN (zOCLN) amino acid sequences and consensus sequence using T-Coffee (see Methods for details). Red underlining of amino acids indicates good sequence similarity. (C) Comparative analysis of the EL1- and EL2-derived sequences of hOCLN with zOCLN. Red underlining of amino acids indicates good sequence similarity. (D) Schematic of the experimental procedure associated with the zebrafish model to test the effect of EL1, EL2 and their scramble peptides on endothelial permeability. Treatment with LPS is used as control for permeability inducing agents. (E) Bar graph of the ratio of leaky endothelium at 3 h post dextran and peptide injection. The experiment was carried out three independent times (nDMSO = 39; nEL1 = 25; nscEL1 = 26; nEL2 = 24; nscEL2 = 23). (F) Bar graph of the ratio of leaky endothelium at 3 h post dextran and peptide injection. The experiment was carried out one time. (nControl = 12; nLPS-50µg/mL = 15; nLPS-100µg/mL = 15). (G) Representative images of zebrafish embryos with an impermeable or leaky endothelium in the tail at 3 h post injection of PBS or LPS, respectively. Scale bar: 100 µm. Data information: In (A, D, E), data are presented as mean ± SD.