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. 2022 Feb 11;221(4):e202009037. doi: 10.1083/jcb.202009037

Figure 2.

Figure 2.

Generation and characterization of tricellulin-deficient epithelial cells. (A) Immunofluorescence images showing anti-tricellulin mAb (green) and anti–ZO-1 mAb (magenta) staining in a co-culture of WT and Tric KO EpH4 cells. Dotted line overlays the border between WT and Tric KO cells, which are indicated by the asterisk. Scale bar: 20 μm. (B) Whole-cell lysates of WT, Tric KO, and hTric-cGFP res EpH4 cells were immunoblotted with the indicated antibodies. Molecular weight measurements are in kD. (C) WT, Tric KO, and hTric-cGFP res EpH4 cells were stained with anti–claudin-3 pAb. Insets are enlarged below. Scale bar: 20 μm. (D) Graph showing the rate of disrupted tTJs. The numbers of closed and disrupted vertices were automatically quantified based on images represented in C. Examples of the processed postanalysis images are enlarged below the graph. Closed tTJs are encircled in blue, and disrupted tTJs, in orange. One-way ANOVA with Tukey’s post hoc analysis; ns, P > 0.05; ****, P < 0.001. (E) WT, Tric KO, and hTric-cGFP res EpH4 cells were co-stained with anti-occludin mAb (green) and anti–ZO-1 mAb (magenta). Insets are shown below. Scale bar: 20 μm. (F) WT cells, three independent clones of Tric KO cells, and three independent clones of hTric-cGFP res EpH4 cells were prepared for TER measurements as described in Materials and methods (means ± SD; n = 6). (G) Graph showing the paracellular flux of FITC-dextran tracer molecules over 2 h (n = 3; P values from one-way ANOVA with Tukey’s post hoc analysis are shown). (H) Cell-surface biotinylation assay in WT and Tric KO EpH4 cells. Cells were treated with sulfo-NHS-SS-biotin from the apical side, fixed, and stained with anti–claudin-3 pAb (green) and Texas Red streptavidin (magenta). Scale bar: 5 μm. (I) Schematic showing the architecture of tTJs in WT and Tric KO cells. Bicellular TJ strands that converge at tricellular contacts are closely associated in WT cells but are disassociated in Tric KO cells. Source data are available for this figure: SourceData F2.