Figure 5.

Extracellular matrix (ECM) coating regulates the expression of cytokeratin (CK)10, CK14 and CK19 in epidermal keratinocytes in vitro. Human basement membrane (BM) extract-derived ECM was used to minic the BM structure in vitro. (A and B) Representative fluorescence images showing the localization of integrin-β4 (green) in (A) human epidermal keratinocytes (HEKs) and (B) human immortalized keratinocyte (HaCaT) cells with or without ECM coating. Nuclei were stained with DAPI (blue). All scale bars represent 25 μm. (C) Western blot analysis of integrin-β4 expression in HEKs and HaCaT cells with or without ECM coating. (D–I) After primary cultured HEKs and HaCaT cells were switched to the medium containing 1.5 mM Ca²⁺ for the indicated time periods, the relative levels of CK10, CK14, and CK19 expression were assessed by real-time qPCR. Ca²⁺ induced the expression of (D and G) CK10 and (E and H) CK14 in a time-dependent manner in the epidermal cell lines, with a peak expression level 24 h after 1.5 mM Ca²⁺ treatment in (D and E) HEKs and (G and H) HaCaT cells. ECM treatment, however, reduced the differentiating responses of keratinocytes associated with Ca²⁺ administration in (F) HEKs and (I) HaCaT cells, and enhanced expression of CK19 at 12 h and 24 h after Ca²⁺ addition. Data are the means ± SD. ^*P<0.05, ^**P<0.01, ns= not significant. (J) Western blot analysis of CK10, CK14, and CK19 expression in HEKs and HaCaT cells with or without ECM coating.