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Fig. 3. Weak cell adhesion between mutant keratinocytes. (A-C) Newborn mutant (Mut) and wild-type control (Wt) keratinocytes were grown in vitro to confluency, and then cultured in high Ca²⁺ medium for 24 hours to induce desmosome formation. The cell sheets were then lifted from the plate and exposed to mechanical stress (Huen et al., 2002). (A) Mutant cell sheets showed increased susceptibility to stress. Each well represents cells from a single pup, i.e. three mutant and three wild-type pups were used. (B) Quantitative evaluation of particles generated in the experiment shown above (average number of particles; n=3; error bar, standard deviation). (C) LDH release into the culture medium of stressed cell sheets. Mutant samples did not show increased cell lysis (LCT, lysis control; wild-type cells were treated with a detergent to release LDH; positive control). (D) Immunofluorescence staining of wild-type and mutant keratinocytes with antibodies against DSP (green) and K14 (red) (DAPI staining of the nucleus, blue). Bars, 50 µm. (E) Electron micrograph of desmosomes formed in newborn wild-type and mutant keratinocytes that were cultured in high Ca²⁺ medium for 24 hours. Morphologically normal desmosomes were formed in mutant keratinocytes. Bar, 0.5 µm. (F) Western blot analysis of newborn keratinocytes cultured as described in A. Detergent-insoluble (junction associated) and detergent-soluble (non-junctional) protein fractions were isolated as described (Cheng et al., 2004) and probed with the antibodies listed. β-Actin or keratin 5 (K5) were used as loading controls.

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