Figure 2.
EpCAM is involved in the barrier function of epithelial cells. (A) Immunostaining of EpCAM in epithelial tissues. Mouse colon and kidney were stained with rat anti-EpCAM mAb (red) and rabbit anti–ZO-1 pAb (green) together with DAPI (blue). Enlarged image of the region in the kidney (yellow dotted rectangle) was shown (right panels). Asterisks indicate lumens. Scale bars, 10 µm. (B) Immunoblotting of epithelial tissue lysates. Lysates from mouse lung, ileum (small intestine), colon, and kidney were analyzed using rabbit anti-EpCAM and mouse anti–β-actin mAbs. Full-length (black arrowhead) and cleaved (gray arrowhead) bands of EpCAM were detected. β-actin served as an internal control to assure that similar amounts of proteins were applied. (C) Immunoblotting of the Ctrl and EpCAM-KO MDCK II cells. HEK293T cells expressing EpCAM-FLAG served as a positive control. Cell lysates were labeled with mouse anti-EpCAM and mouse anti–β-actin mAbs. (D and E) Immunostaining of the Ctrl (upper panels) and EpCAM-KO (lower panels) MDCK II cells. Cells were stained with mouse anti-EpCAM mAb (D) or rabbit anti–claudin-7 pAb (E; red) together with rat anti–ZO-1 mAb (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 TJs, respectively. Scale bar, 10 µm. (F) TER measurement of EpCAM-KO cells. n = 13 (Ctrl and KO#1) and 10 (KO#2 and KO#3). The values of KO cells were compared with those of Ctrl cells at each time point using two-tailed Welch’s t test with Bonferroni’s correction (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; actual P values are shown for 0.05 < P < 0.001). (G) FFEM analysis of EpCAM-KO cells. Scale bar, 200 nm. (H) Quantification of the frequency of TJ strand branching points in the EpCAM-KO cells. Total length of TJ strands examined was 122 µm (Ctrl) and 156 µm (EpCAM-KO#1). Error bars indicate 95% confidence intervals. ***, P <0.001 (exact Poisson test). (I) Distribution of horizontal TJ strand number in the EpCAM-KO cells. Blue lines indicate mean ± SD. n = 259 (Ctrl) and 302 (EpCAM-KO#1). ***, P < 0.001 (two-tailed Welch’s t test). (J) Predicted TER values using simplified TJ strand network models based on the quantification data of TJ strand network complexity. Source data are available for this figure: SourceData F2.

EpCAM is involved in the barrier function of epithelial cells. (A) Immunostaining of EpCAM in epithelial tissues. Mouse colon and kidney were stained with rat anti-EpCAM mAb (red) and rabbit anti–ZO-1 pAb (green) together with DAPI (blue). Enlarged image of the region in the kidney (yellow dotted rectangle) was shown (right panels). Asterisks indicate lumens. Scale bars, 10 µm. (B) Immunoblotting of epithelial tissue lysates. Lysates from mouse lung, ileum (small intestine), colon, and kidney were analyzed using rabbit anti-EpCAM and mouse anti–β-actin mAbs. Full-length (black arrowhead) and cleaved (gray arrowhead) bands of EpCAM were detected. β-actin served as an internal control to assure that similar amounts of proteins were applied. (C) Immunoblotting of the Ctrl and EpCAM-KO MDCK II cells. HEK293T cells expressing EpCAM-FLAG served as a positive control. Cell lysates were labeled with mouse anti-EpCAM and mouse anti–β-actin mAbs. (D and E) Immunostaining of the Ctrl (upper panels) and EpCAM-KO (lower panels) MDCK II cells. Cells were stained with mouse anti-EpCAM mAb (D) or rabbit anti–claudin-7 pAb (E; red) together with rat anti–ZO-1 mAb (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 TJs, respectively. Scale bar, 10 µm. (F) TER measurement of EpCAM-KO cells. n = 13 (Ctrl and KO#1) and 10 (KO#2 and KO#3). The values of KO cells were compared with those of Ctrl cells at each time point using two-tailed Welch’s t test with Bonferroni’s correction (ns, P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001; actual P values are shown for 0.05 < P < 0.001). (G) FFEM analysis of EpCAM-KO cells. Scale bar, 200 nm. (H) Quantification of the frequency of TJ strand branching points in the EpCAM-KO cells. Total length of TJ strands examined was 122 µm (Ctrl) and 156 µm (EpCAM-KO#1). Error bars indicate 95% confidence intervals. ***, P <0.001 (exact Poisson test). (I) Distribution of horizontal TJ strand number in the EpCAM-KO cells. Blue lines indicate mean ± SD. n = 259 (Ctrl) and 302 (EpCAM-KO#1). ***, P < 0.001 (two-tailed Welch’s t test). (J) Predicted TER values using simplified TJ strand network models based on the quantification data of TJ strand network complexity. Source data are available for this figure: SourceData F2.

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