Fig 1: Mice deficient of uPAR display improved intestinal epi-barrier function. (a). Workflow of the monolayer assay derived from primary murine intestinal organoids from WT (Plaur +/+) and uPAR deficient (Plaur -/-) mice. Monolayer breakdown was triggered by simultaneous murine TNF-α and IFN-γ treatment (10 ng/ml each). 3 donors of each genotype were used in the monolayer assay. (b). Barrier function after TNF-α and IFN-γ induced damage in monolayers derived from WT or uPAR deficient murine intestinal organoids (n = 9/treatment group). TEER and FITC-Dextran permeability were assessed. Stim, murine TNF-α and IFN-γ treatment for 48 hours. (c). Barrier formation in WT and uPAR deficient monolayers was monitored (3 donors/genotype, D1, D2 and D3). (d). Localization of ZO-1 (red) and JAM-A (magenta) in the murine organoid-derived monolayers before (-) and after (stim) barrier damage. Nuclei are visualized with DAPI (blue) in the bottom panel. Bright field images of 3D organoids and 2D monolayers are presented on the top panel. Relative intensity is presented on the right (n = 5/group, normalized to WT controls). ZO-1 and JAM-A protein levels before and after barrier damage are presented in Figure S6e. (e). Workflow of the DSS-colitis study. (f). Histopathology assessment of naïve WT controls (n = 8), WT (n = 17) and uPAR deficient mice (n = 18) after DSS-induced colitis. 4 sections from each animal were scored in a double-blinded fashion and average scores are presented on the right. See Methods for more details. Representative images were presented with the corresponding scores. (g). Localization of ZO-1 (red) and JAM-A (magenta) in colonic sections from naïve controls, WT or uPAR KO mice after DSS treatment. Nuclei are visualized with DAPI (blue). Regions with disrupted tight junction are marked with white arrows. Relative intensity is presented below (naïve: n = 3, WT or KO DSS treated groups: n = 4). (h-i). Gene expression profiling (shown as Heatmap, h) of distal colon tissue from the DSS colitis study comparing WT and uPAR KO mice. Genes displayed are all DSS-induced genes in WT mice (FC >1.5, p < 0.05, total = 192 genes). Detailed expression levels of S100a8, S100a9, Spp1, Mmp13, Il6 and Il1b from the Nanostring analysis of individual animals is shown (i). All data are shown as mean ± SD and are analysed using 2-way ANOVA followed by Šidák's multiple comparison test (b, d) or 1-way ANOVA followed by Dunnett's multiple comparison test (c, f, g, i).