Both the trans-interaction and ZO-1 binding of claudin and JAM-A are important to support apical junction integrity. (A) Schematic illustration of the constructs used in the study. Claudin-1[F147A] is a mutant that inhibits the trans-interaction and strand formation, while claudin-1[ΔYV] lacks the PDZ-binding motif required for the interaction with ZO-1. JAM-A[ΔDL1] inhibits the cis-dimerization and trans-interaction, while JAM-A[ΔLV] lacks the PDZ-binding motif required for the interaction with ZO-1. (B–D) ZO-1 staining of claudin/JAM-A KO cells rescued with claudin-1 full-length (FL; B), claudin-1[F147A] (C), or claudin-1[ΔYV] (D). While claudin-1 full-length completely restored the junction continuity (B), claudin-1[F147A] (C) and claudin-1[ΔYV] (D) failed to rescue the junction breakage phenotype (asterisks). (E–G) ZO-1 staining of claudin/JAM-A KO cells rescued with JAM-A full-length (FL; E), JAM-A[ΔDL1] (F), or JAM-A[ΔLV] (G). JAM-A full-length completely rescued the junction breakage phenotype (E). JAM-A[ΔDL1] suppressed the formation of large gaps but focal discontinuity of cell junctions was frequently observed (F′, arrow). JAM-A[ΔLV] failed to rescue the junction breakage phenotype (G′, asterisks). (H) Quantification of the junction breakage phenotype. Data represent the numbers of endpoints of ZO-1 staining per unit area and are presented as mean ± SD (n = 9). ***P < 0.0005, compared with claudin/JAM-A KO cells by one-way ANOVA with Tukey post hoc test. Scale bar: 10 μm.