Figure 4.

CAST Knockdown Decreases Intercellular Adhesion and Increases Desmoplakin Abundance

(A–C) HaCaT cells transfected with a pool of NTP siRNA (A) or CAST siRNA (B) were stained with an anti-calpastatin (green) antibody and DAPI (blue) as a nuclear marker. Total protein from HaCaT cell lysates after transfection with NTP siRNA or CAST siRNA were incubated with an anti-calpastatin antibody (C). GAPDH was used as loading control. A significant reduction in calpastatin protein abundance can be observed in the CAST KD cells compared to NTP cells, both by immunocytochemistry and immunoblotting.

(D–G) CAST KD (F and G) and NTP (D and E) cells were subjected to cyclic stretching for 0 hr (unstretched; D, F) and 4 hr (E, G) followed by staining with an anti-keratin 14 antibody. Stretching of the keratin filaments can be observed after 4 hr mechanical stress in the NTP cells (arrows) compared to unstretched monolayers. In the CAST KD cells, breakage of these filaments together with widening of the intercellular spaces was observed independent of mechanical stress (asterisks).

(H and I) Immunohistochemistry with an anti-desmoplakin antibody on skin sections from individual 2 revealed an upregulation and change in localization of this protein from a membranous appearance (H) to a both membranous and cytoplasmic appearance (I) in comparison to control.

The scale bars in each image represent 20 μm.