Focal breakage of apical junctions in claudin/JAM-A KO cells. (A) ZO-1 staining of MDCK II cells. A continuous chicken-wire pattern was observed. (B) ZO-1 staining of claudin/JAM-A KO cells. Focal junction breakage was evident, wherein ZO-1 staining was discontinuous (B′, arrow) or large gaps in the ZO-1 network were observed (B″, asterisk). (C) Quantification of the junction breakage phenotype in claudin/JAM-A KO cells. The 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 by one-way ANOVA with Tukey post hoc test. (D) ZO-1/occludin/ZO-2 triple staining of MDCK II cells. A continuous chicken-wire pattern was observed for all markers. (E) ZO-1/occludin/ZO-2 triple staining of claudin/JAM-A KO cells. Junction breakage (asterisks) was observed for all markers. (F) ZO-1/afadin/E-cadherin triple staining of MDCK II cells. A continuous network of ZO-1, afadin, and E-cadherin was evident in MDCK II cells. (G–J) ZO-1/afadin/E-cadherin triple staining of claudin/JAM-A KO cells. Junction breakage was observed for ZO-1 (G, asterisk) and afadin (G′, asterisk). Discontinuity of E-cadherin staining was observed at the apical AJs (G″, asterisk; I, arrow), while continuous E-cadherin staining was maintained at the lateral cell contacts (H″, J, arrow; asterisk indicates the region with junction breakage). Maximum intensity projections of apical (G and I) and lateral (H and J) confocal sections are shown. (K) ZO-1/desmoplakin double staining of MDCK II cells. Desmoplakin was localized at a dotted belt-like structure at the apical region and formed puncta at the lateral membrane. (L–N) ZO-1/desmoplakin double staining of claudin/JAM-A KO cells. The apical dotted belt-like structure of desmoplakin was disrupted at the large gaps of ZO-1 staining (L′, asterisk), while continuous desmoplakin staining was retained at the lateral membrane and small ZO-1 discontinuities (M and N, arrowheads). Scale bars: 10 μm.