FIGURE 4:

Cldn3 and 4 mediate the function of Grhl2 to enlarge a central lumen. (A) Cldn3 and 4 in HPPL-Grhl2 are localized at TJs. Cldn3 (red) expressed in HPPL-Grhl2 is colocalized with ZO1 (green; 5–8). Cldn4 (red) expressed in HPPL-Grhl2 is colocalized with ZO1 (green) in addition to relatively strong expression at the basolateral membrane (13–16). Cysts derived from HPPL and HPPL-Grhl2 were stained with anti-ZO1 and anti-Cldn3 or 4 antibodies. Nuclei were counterstained with Hoechst 34580 (blue). (B, C) Inhibition of Cldns by the C-terminal peptide derived from C-CPE decreases the size of a central lumen. Pictures for representative cysts are shown in B. The central lumen of cysts in the presence of CPE peptide is normally surrounded by F-actin bundle (green) but is remarkably smaller compared with the control. (C) Dot plot with bars of mean ± SEM for a representative culture, indicating that the lumen becomes smaller in the presence of CPE peptide with statistical significance. HPPL and HPPL-Grhl2 were cultured in the presence of 100 μg/ml GST-C-CPE or GST-C-CPE313 (a deletion mutant of C-CPE lacking the capability to bind to Cldns). The diameter of lumen was measured for >50 cysts per culture. Experiment was repeated four times independently. (D, E) Overexpression of Cldn3, but not Cldn4, induces the enlargement of a central lumen. (D) Representative pictures of cysts derived from HPPL, HPPL-Cldn3, and HPPL-Cldn4. Cldn3, but not Clnd4, enlarged the central lumen of a cyst. (E) Dot plot with bars of mean ± SEM for a representative culture, indicating that Cldn3 increases the size of lumen with statistical significance. HPPL, HPPL-Cldn3, and HPPL-Cldn4 were cultured under 3D conditions for 7 d. The diameter of lumen was measured for >50 cysts per culture. Experiment was repeated four times independently.