Fig 2: Localization of claudins at TJs between control and their respective knockout cells.(A–E) Immunofluorescence analysis of claudins and ZO-1 or ZO-3 in MDCK II cells transfected with the TALEN constructs for claudin-1, -2, -3, -4, or-7 gene knockouts. After transfection, cells were subcultured on filter inserts for 4 days before the analysis of claudin-2 and -3 (B and C), and for 2 days before the analysis of claudin-1, -4, and -7 (A, D, and E). (F) Immunofluorescence analysis of claudin-2 and ZO-1. TALEN constructs for the claudin-2 gene knockout were transfected into MDCK II cells, which were subcultured on filter inserts for 2 days before analysis. (G) Immunofluorescence analysis of claudin-2 and ZO-1 in a co-culture of wild-type and claudin-2 expressing MDCK I cells. Cells were cultured on filter inserts for 4 days before analysis. Scale bar = 10 µm.

Fig 3: Effects of ML-7, a specific MLCK blocker, were studied after TNFa incubation using TEER measurements and Western blot analysis. (A) After 48 h, the groups treated solely with TNFa showed a significant difference in TEER compared to controls and cells treated with TNFa and ML-7 (one-way ANOVA, ** p < 0.01, *** p < 0.001; n = 7–29). (B) Representative Western blot bands and (C) densitometry after 48 h with TNFa in the presence or absence of ML-7 revealed that the MLCK blocker prevented the TNFa-induced decrease in claudin-1 and -3, while the decrease in occludin was not affected. Furthermore, the TNFa-induced increase in TNFR-1 is also inhibited by ML-7. Data are presented as mean ± SEM (* p < 0.05, ** p < 0.01; n = 4).

Fig 4: Effects of ZO-1 knockout on the localization and expression levels of claudins.(A) Effects of ZO-1 knockout on the localization of claudins. Control and ZO-1 knockout cells of clone 1 were co-cultured on filter inserts. Signal intensity of claudins on lines shown in confocal microscopic images (arrows) were analyzed. Claudin-2 fluorescent signal at TJs was increased but claudin-1 and -7 signals at TJs were reduced in ZO-1 knockout cells. Scale bar, 10 µm. (B) Immunoblots of claudins in control MDCK II cells and ZO-1 knockout clones. Similar expression levels of claudins were observed in control and knockout cells.

Fig 5: Confocal laser scanning immunofluorescence microscopy of (A) claudin-1 (green) and ZO-1 (red), as well as (B) claudin-3 (green) and occludin (red), nuclei were stained in blue (DAPI). IPEC-J2 monolayers were treated for 48 h with TNFa in the presence or absence of the specific MLCK blocker ML-7. (B) The yellow signal in the merged pictures represents the colocalization of claudin-3 and occludin (scale bar: 20 µm; n = 4; representative images).