Fig. 4

CRISPR/Cas9 knockout of occludin in Caco-2 cells reveals partial downregulation of ZO-1. Three individual occludin knockout clones of Caco-2 cells were analysed. Western blotting confirms the presence of occludin in Caco-2 wild-type (wt) cells and absence in the knockout cells (a). Phase contrast microscopy of Caco-2 wt cells (top) and occludin knockout cells (bottom) revealed only slight phenotypic differences (b). Immunofluorescence staining of Caco-2 wt (top) and occludin knockout cells (bottom) stained for ZO-1 (green), presence of the HDR-plasmid (red) and DNA in the nuclei by DAPI (blue). The red fluorescence derives from the CRISPR–Cas9 plasmid, which was used for stable transfection (c)