Fig. 5. Establishment of a mouse keratinocyte cell line, carrying a conditional allele of the ß4 gene. (A) Phase-contrast image of subconfluent NMK-1(+) and (–) keratinocytes showing their morphology. (B) Immunoblot analysis of the expression levels of p53 and different epithelial and mesenchymal markers in the NMK-1(+) and (–) cell lines. RAC-11P cells (Sonnenberg et al., 1993) and mouse embryonic endothelial cells (MEEC) (Larsson et al., 2001) are used as control cell lines. (C) FACS analysis of the levels of 6, ß4, ß1, 2, 5 and v integrin subunits expressed by NMK-1(+) and (–) cells. (D) Immunoprecipitation analysis of surface-labeled components from NMK-1(+) and (–) cells with antibodies specific for a range of integrin subunits. (E) NMK-1(+) and (–) keratinocytes adhered equally well to Ln-5 and fibronectin. (F,G) Detachment assays based on resistance to trypsin treatment (F) and centrifugal force (G) demonstrate the importance of 6ß4 in strengthening the adhesion. (H) Indirect immunofluorescence analysis of hemidesmosomal protein localization in NMK-1 cells visualized by confocal microscopy. Primary antibodies are against proteins specified in the left lower corner of each image and colors are coded according to FITC or Texas Red secondary antibodies. The colocalization of the different hemidesmosomal components indicates that these cells form type I HDs in culture. (I) Ultrastructural analysis of NMK-1(+) and (–) cells (upper and lower panels, respectively) further confirms the presence of HDs in NMK-1(+) cells whereas they are lost in NMK-1(–) population. (J) Wound healing assay shows that the motility of NMK-1(–) cells is increased compared with those of NMK-1(+). In the bar graph, results are expressed as the unadjusted means ± s.d. of four separate experiments with six replicates each (P<0.001).