GRHL2 can be detected in the nuclear fraction of normal human keratinocytes in culture (but is subsequently lost in cell senescence).23 We cultured keratinocytes from two of the skin biopsies (ED-02 VI-3 and ED-02 VI-5) by standard methods and used these cells to examine GRHL2 expression and GRHL2 localization (see Supplemental Data for methods); both were found to be reduced (Figures 2D and 2E). We then isolated primary keratinocytes from one of the affected individuals (ED-02 VI-5) and immortalized these cells at passage 1 (see Supplemental Data for methods). The phenotype of these cells was assessed by confocal microscopy. GRHL2 mutant cells showed a less cuboidal, elongated phenotype and failed to form intact cell junctions, as seen in control immortalized keratinocytes. Notably, there was a reduction in cell membrane labeling for E-cadherin (adherens junctions) and zona-occludens-2 (tight junctions) (Figure S5). GRHL2 staining in control keratinocytes was seen both at cell-cell contact areas and within the nucleus (Figure 3A), whereas in mutant cells, the signal was not at the periphery and instead showed a fragmented punctate nuclear localization (Figure 3B). To assess the effects of the mutations on keratinocyte cell function, we also performed assays of cell adhesion and de-adhesion (see Supplemental Data for methods). No differences were noted for cell adhesion between mutant and control cells (Figure 3C), but mutant cells detached from fibronectin much faster than normal human keratinocyte controls after exposure to trypsin (Figure 3D).