Downregulation of E-cadherin by ET-1 in melanoma cells (SKMEL28) and melanocytes (71201L, FM2030). Lysates normalized for protein content were analyzed for E-cadherin protein levels by immunoblot analysis. (a) Cells were stimulated with 10 nM ET-1 for the indicated times. Un, unstimulated controls. ET-1 was removed after 3 hours, culture medium replaced with ET-1–free medium, and cells harvested at 40 hours (lower-right panel). (b) N-cadherin, CD44, and ICAM-1 are not affected by ET-1. (c) E-cadherin Northern blot of total RNA isolated from SKMEL28 cells. GAPDH serves as an internal control. Identical results were obtained using melanocytes (data not shown). (d) ETB receptor antagonist BQ788 blocks E-cadherin downregulation by ET-1. Cells were incubated with either ETA receptor antagonist BQ123 or ETB receptor antagonist BQ788 as indicated. (e) E-cadherin downregulation by ET-1 is dose responsive. Lanes 2–4 are labeled with the nM concentrations of ET-1 used. Lane 5, 10 nM ET-3 was used. (f) E-cadherin downregulation by UVB occurs via an ET-1/ETB–dependent pathway. Conditioned medium from UV-irradiated keratinocytes (UV-KCM) was used to stimulate SKMEL28 cells for 40 hours (left panel). First lane, KCM from unirradiated control (Un). Last lane, cells were pretreated with ETB receptor antagonist BQ788. Cells were stimulated for 40 hours with conditioned medium from keratinocytes stimulated with IL-1α (middle panel). Cells were stimulated with purified recombinant purified IL-1α (right panel). Identical results were obtained using melanocytes (data not shown). (g) ET-1 ELISA assay of UV-KCM and IL-1-KCM. Results shown are representative of at least four independent experiments. E-cad, E-cadherin.