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Fig. 5 ZnUMBA detects transient local barrier defects in MDCK II cultured epithelial cells. (A) ZnUMBA of WT cells and Cldn2-KO cells. GFP–nls-labeled WT MDCK II cells and Cldn2-KO MDCK II cells were mixed and co-cultured on the bottom surface of the filter. Images before (left) and after (middle) FZ3 addition are shown. The Fire lookup table (LUT) has been applied using ImageJ. Right panel is the magnified view of the region outlined by a rectangle in the middle panel. Note that the cell–cell junctions between WT cells labeled with nuclear GFP have higher signal compared to those between Cldn2-KO cells. Scale bars: 20 µm. (B,C) ZnUMBA of Ang1-KO cells in the Cldn2-KO background. GFP–nls-labeled Ang1/Cldn2-dKO MDCK II cells and Cldn2-KO MDCK II cells (B), or Ang1/Cldn2-dKO MDCK II cells and mCherry–nls-labeled Cldn2-KO MDCK II cells (C), were mixed and co-cultured. Fire (B) and Gem (C) LUTs from ImageJ have been applied to the FZ3 channel. mCherry signal is shown pseudocolored in blue (C). Boxes indicate regions shown as magnified views in the right-hand panels. Note that tricellular junctions between Ang1/Cldn2-dKO cells (green arrows) have higher signal than bicellular junctions. Scale bars: 20 µm. Images in A–C are representative of four experiments.