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Figure 8


Fig. 8. Knockdown of CASK leads to epidermal hyperproliferation in organotypic cultures. (A) Protein extracts from epidermal tissue were isolated from organotypic cultures, and prepared with control-siRNA- and CASK-siRNA-transfected HaCaT cells after 7 days at the air-liquid interface. Immunoblotting was performed using antibodies against CASK (112 kDa), PCNA (30 kDa), Myc (64 kDa), involucrin (120 kDa) and filaggrin (40 kDa), actin (42 kDa) was used as an internal control. Notice the reduced CASK protein level in CASK-siRNA-transfected epidermal extracts concomitant with the upregulation of PCNA and Myc. (B) H&E histology and immunofluorescence staining for CASK in control and CASK-knockdown HaCaT raft cultures. H&E histology shows a thicker epidermal morphology in the CASK-knockdown 3D cultures, consisting of several layers compared with the control-siRNA-transfected rafts. (C) Expression of keratins, proliferation and differentiation markers, and basal integrins in siRNA-transfected raft cultures. Notice the presence of Ki67-positive cells and H3-P-positive mitotic cells in the CASK-knockdown skin model, in both basal and suprabasal layers of the epidermis. Involucrin and filaggrin are expressed at similar levels in the suprabasal and differentiating layers of the epidermis. CASK knockdown skin model shows integrin expression also in lateral and apical membranes of the basal cells. All protein staining are shown in green and DAPI nuclear staining is shown in blue. Scale bars, 50 µm.





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