KO of EpCAM and c laudin-7 genes. (A) Genomic structure and gene-KO strategies of EpCAM. gRNA sequences used for genome editing (red) are shown. The deleted region of the EpCAM gene contains the first exon with a start codon. (B) Screening strategy of EpCAM-KO cells. In the WT cells, the F1-R2 fragment is not amplified because of the high guanine-cytosine content (GC-rich) region. (C) Genomic PCR of Ctrl and EpCAM-KO clones. (D–F) Immunostaining of the Ctrl cells (top panels) and EpCAM-KO cells (bottom panels). Cells were stained with anti–claudin-1 (D), anti–claudin-3 (E), or anti–claudin-4 (F) (red) together with anti–ZO-1 (green) and DAPI (blue). Gray arrowheads on the side of the top views indicate the location where the side-view section was made, and the black arrows and arrowheads below the side-view panels indicate the locations of the bicellular and tricellular junctions, respectively. Scale bars, 10 µm. (G) Enlarged image of the predicted structure of EpCAM–claudin-7 complex. Yellow arrow indicates the putative site of second cleavage, which corresponds to the band in Fig. 4, A and B, indicated with blue arrowheads. (H) Genomic structure and gene-KO strategy of claudin-7 (CLDN7). (I) Screening strategy of claudin-7-KO cells. (J) Genomic PCR of the Ctrl and claudin-7-KO cells. (K) Immunoblotting of claudin-7-KO cells. (L) Tracer flux assay of the claudin-7-KO cells using 10-kD FITC-dextran. N = 6. Statistical significance compared with the Ctrl cells was evaluated by two-tailed Welch’s t test with Bonferroni’s correction (**, P < 0.01; ***, P < 0.001). Source data are available for this figure: SourceData FS2..