Fig. 4

IFN-γ activates macrophages through Stat1 to impair the barrier function of CHPEs. (a) Schematic diagram of two models include CHPEs co-cultured with macrophages (①) and macrophage-conditioned medium (②). ① shows the culture of CHPE and macrophages in the Transwell system. In the migration assay, macrophages were seeded in the upper chamber of the Transwell insert, while CHPE were seeded in the lower chamber (culture plate). In the leakage assay, the arrangement was reversed. ② shows the co-culture of CHPE and macrophages with conditioned medium supernatant. ① represents the co-culture models prefixed with “Co-“, and ② represents the models prefixed with “S-” (b) Representative images of macrophage migration assay. Scale bars, 100 μm. Quantification of cells on the back of the chamber (n = 3). Unpaired T tests were applied. ** P < 0.01. (c) Representative immunoblots of Occludin and β-Catenin protein levels in CHPEs under two co-culture conditions. (d) Quantification of fluorescence intensity in the lower chamber medium for the two co-culture models (n = 4). Unpaired T tests were applied. ** P < 0.01 and * P < 0.05. (e, f) Representative IF staining images and quantification of Occludin and β-Catenin fluorescence intensity in CHPEs co-cultured with macrophages (e) and macrophage-conditioned medium (f), (n = 3–4). Scale bars, 50 μm. Unpaired T tests were applied. ** P < 0.01 and * P < 0.05. (g) Representative IF images of CD68 staining in macrophages. Scale bars, 50 μm. (h) qPCR analysis of stat1, il1β, nos2, and tnfα mRNA expression in macrophages (n = 6). One-way ANOVA was applied. *** P < 0.001. (i and j) Representative IF staining images (i) and quantification (j) of Occludin and β-Catenin fluorescence intensity in CHPEs (n = 4). Scale bars, 50 μm. One-way ANOVA tests were applied. ** P < 0.01 and * P < 0.05. (k) Representative immunoblots of Occludin and β-Catenin protein levels in CHPEs. (l) Quantification of fluorescence intensity in the lower chamber medium for the conditioned medium co-culture model (n = 4). One-way ANOVA tests were applied. *** P < 0.001