Figure 4.

Organotypic Cultures

(A) Full skin models were generated with NTP and CSTA KD. Loss of cystatin A was shown by immunoblot analysis in protein extracts from skin models and by immunostaining against cystatin A.

(B) Hematoxylin and eosin staining showing completed epidermal differentiation, hyperkeratosis, and parakeratosis, and a disturbance of basal epidermal architecture in CSTA KD organotypic culture compared to NTP organotypic cultures. The scale bar represents 50 μm.

(C) Immunostaining against keratin 14 (upper panel) and E-cadherin (lower panel) demonstrated grossly disorganized structure, widening of intercellular spaces (arrows), and partial breakdown of keratin intermediate filaments in basal and lower suprabasal layers of disease models depleted of cystatin A. The scale bar represents 20 μm. SC is an abbreviation of stratum corneum.

(D) Barrier function appears to be largely maintained in the CSTA KD organotypic culture in a Lucifer Yellow dye penetration assay (shown in green) compared to the NTP organotypic culture and in contrast to the loss of barrier function observed in the corneodesmosin knockdown (CDSN KD) organotypic culture, which is a model of the generalized peeling skin disease. The scale bar represents 100 μm.