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) ET_B receptor antagonist BQ788 blocks E-cadherin downregulation by ET-1. Cells were incubated with either ET_A receptor antagonist BQ123 or ET_B 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/ET_B–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 ET_B 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.