sPmel17 Secreted by Ultraviolet B-Exposed Melanocytes Alters the Intercellular Adhesion of Keratinocytes

Figure 1

MCs achieve the phenotypes of melanogenesis and migration following UVB exposure. Primary human epidermal MCs were isolated from juvenile foreskin tissues, treated with or without 30 mJ/cm² UVB and then cultured for one additional day. (a) qRT-PCR was performed to measure the expression levels of melanogenesis-associated mRNAs (MITF, TYR, TYRP1, DCT, and Pmel17) and the migration-related MCAM in MCs exposed or unexposed to 30 mJ/cm² UVB. The results were normalized to the housekeeping gene GAPDH. The data represent from 3 independent experiments. ; . (b) MCs were lysed in extraction buffer, after which specific antibodies against human MITF, TYR, TYRP1, DCT, Pmel17, and MCAM were used in western blotting to examine their corresponding protein levels. Protein loading variations were determined by immunoblotting with an anti-GAPDH antibody. Representative blots are shown on the left. The histograms show the densitometric quantification of data with from 3 independent experiments. ; . (c) Supernatants were collected from UVB-exposed and from unexposed MCs, and the protein concentration in each supernatant was measured using the BCA assay, after which the supernatants were further concentrated by lyophilization as described in Materials and Methods. Levels of sPmel17 in the supernatants from UVB-exposed or UVB-unexposed MCs detected by western blotting are shown (upper panel, right) (). An equal amount of total protein (20 μg) of each supernatant was loaded per lane and was separated by SDS-PAGE. A gel stained with Coomassie blue solution is shown (left panel). The black arrowhead indicates the band at 100 kDa of presumed sPmel17. (d) Representative images for cell migration assessed by the Transwell assay (scale  μm). Histograms show the number of cells passing through the insert membrane. The data represent from 3 independent experiments. .

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