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. 2023 May 19;222(7):e202208065. doi: 10.1083/jcb.202208065

Figure 8.

Figure 8.

CGN binding to NM2B and NM2 activity is required to maintain TJ membrane tortuosity in MDCK cells. (A–C) IF microscopy localization of β-catenin (red) and CGN (green) in WT MDCK cells (A), of occludin (red) in WT, CGN-KO, CGNL1-KO, and CGN/CGNL1-double-KO (dKO) MDCK clonal cell lines (B), and quantifications of membrane tortuosity (ZI) in WT, CGN-KO, CGNL1-KO, and CGN/CGNL1-double-KO cells (C). Dots show replicates (n = 220) and bars represent mean ± SD. Statistical significance was determined by one-way ANOVA with post hoc Dunnett’s test (****P < 0.0001). (D–G) IF microscopy analysis of double-KO cells rescued either with full-length (FL) CGN (D, left panels) or CGNL1 (D, right panels), or with C-terminally truncated CGN (E, left panels) or C-terminally truncated CGNL1 (E, right panels), or GFP (F), and quantifications of ZI in CGN/CGNL1-double-KO cells rescued with the indicated constructs (G) were measured with TJ marker ZO-2. Lines in ZO-2 channel trace membranes to highlight tortuosity. (−) and (+) refer to the same cell line without and with exogenous expression of rescue construct. Dots show replicates (n = 60–87) and bars represent mean ± SD. Statistical significance was determined by unpaired Mann–Whitney’s test, ***P ≤ 0.001. (H–K) IF microscopy localization (H–J) of CGN (green, to detect WT vs. double-KO cells) and the TJ marker ZO-2 (red) either in WT cells (H), or in double-KO cells rescued with FL CGN (I), or FL cCGNL1 (J), and quantification of ZI (K) in the indicated cell lines (WT, double-KO, rescue, and treatments with either DMSO or blebbistatin [blebbi]). In H–J, the panels on the left show cells treated with DMSO (negative control) and the panels on the right indicate cells treated with blebbistatin (4 h, 50 μM). White lines and arrows/arrowheads trace membranes to highlight the shape of the TJ membrane. Arrowheads indicate junctions in double-KO cells. Scale bars = 10 μm. Dots in K show replicates (n = 50–78) and bars represent mean ± SD. Statistical significance was determined by unpaired Mann–Whitney’s test, ***P ≤ 0.001.