WNT5A increases secretion of IL-6, IL-8 and VEGF in cell culture supernatants. (A) Western blot of endogenous WNT5A protein expression in melanoma cell lines Mewo, SkMel28, A2058, A375 and HTB63. Anti-β-actin was used as loading control. Picture is representative of at least 3 experiments. (B) Mewo cells treated with carrier (0.1% BSA in PBS; Ctrl) or 0.2 μg/ml rWNT5A for 3 h. A set of inflammation cytokines were measured using BD Cytometric Bead Array (CBA) and both IL-6 and IL-8 were produced upon a short (3 h) rWNT5A stimulation. (C) IL-6 levels in Mewo cell culture supernatants were measured using IL-6 Elisa. Mewo cells were treated with 0.2 μg/ml rWNT5A or carrier for 3, 6, 12, 24 and 48 h and IL-6 in supernatants was measured. The bar graphs show average fold increase as compared to carrier (ctrl) for each time point indicated. The experiment was performed at least 4 times. Error bars represent SEM. (D) Cells from experiment 1C were used for IL-6 RT-QPCR. The experiment was performed at least 3 times. Error bars represent SD. (E) Supernatants from 1C were used to measure VEGF levels after WNT5A treatments. Error bars represent SEM. (F) Cells from 1C were used for VEGF RT-QPCR. The experiment was performed 3 times. Error bars represent SD. * = p <0.05 by ANOVA test.