Figure 7

Palmitoylated claudin-7 supports the generation of EpIC (a) Confocal microscopy of HEK-EpC-cld7 and HEK-EpC-cld7^mPalm cells stained with anti-EpIC and -cld7; single fluorescence staining and overlays are shown (scale bar: 10 μm) and WB of lysates of PMA-stimulated transfected HEK cells with anti-EpIC; EpIC and EpC bands are indicated. (b) Confocal microscopy of HEK-EpC-cld7 and HEK-EpC-cld7^mPalm cells stained with anti-presenilin2 and -cld7 (single fluorescence and overlays, scale bar: 10 μm); sucrose gradient fractions of lysates of PMA-stimulated HEK-EpC-cld7, HEK-EpC^mAG-cld7 and HEK-EpC-cld7^mPalm cells were immunoprecipitated with anti-presenilin2, precipitates were blotted with anti-EpC, anti-cld7 and anti-presenilin2. (c) WB of presenilin2 and β-catenin in PMA-stimulated transfected HEK cells. (d) Flow cytometry analysis of mesenchymal proteins in PMA-stimulated transfected HEK cells; the mean fluorescence index (% stained cells x mean intensity of staining ± SD; three assays) and representative examples are shown (mean % stained ± SD, mean intensity ± SD; triplicates and representative examples); significant differences between HEK-cld7 versus HEK-EpC-cld7, HEK-EpC^mAG-cld7 and HEK-EpC-cld7^mPalm cells: *. (e) Representative examples of FGF, vimentin and N-cadherin co-localization with cld7 (confocal microscopy, scale bar: 10 μm). Palmitoylated cld7 facilitates the generation of EpIC by associating with presenilin-2 in GEM. EpIC generation is accompanied by a slight upregulation of mesenchymal protein expression.