Fig. 2

Trametinib plus CuET reduces melanoma cell viability in 2D and 3D BRAF WT melanoma cell models. A Cropped Immunoblots of MAPK pathway activity (phospho-ERK1/2^{Thr202/Tyr204} and phospho-MEK1/2^Ser217/221) in the BRAF WT melanoma cell lines SKMEL23 and SKMEL113 after treatment with trametinib (10 nM), CuET (125 nM) or their combination for 6 hours. β-Actin served as the loading control. B Percent viability reduction values (y-axis: Effect_{125 nM CuET}) in response to 125 nM CuET (green symbols) and its combination with 10 nM trametinib (red symbols) in BRAF WT melanoma cells are shown together with corresponding EC₅₀ values (x-axis). Dashed lines represent the mean values of all data points. Data were calculated from cell viability assays (MUH assay) of BRAF WT melanoma cells after 72 hours of treatment. Effect values (100 % corresponds to a complete loss of cell culture viability) and EC₅₀ values (nM) were determined with a dose-response curve using GraphPad Prism. (n = 3 independent experiments; mean values). C Cell viability assays of four BRAF WT models that were treated with increasing concentrations of Cu²⁺ (up to 500 nM), ET (up to 500 nM) and CuET (up to 500 nM) alone or in combination with 10 nM trametinib for 72 hours. Viability measurements were normalized to the untreated control (n = 3 independent experiments measured in hexaplicates; mean ± SD). Dotted lines represent the effect of 10 nM trametinib on cellular viability. D Clonogenic growth assay of BRAF WT melanoma cells after 14 d of treatment with trametinib (10 nM), CuET (125 nM) or their combination. Representative results of crystal violet staining are shown. Three independent experiments were quantified, and differences between the treatment groups were analyzed by two-way ANOVA with subsequent Tukey’s multiple comparisons test. E Invasive growth of 3D spheroids generated from BRAF WT melanoma cells (SKMEL23, SKMEL113, TÜMEL62-1 and TÜMEL173). Spheroids were embedded into a type I collagen matrix and treated with 10 nM trametinib, 125 nM CuET or their combination for 7 days. Light microscopic images were taken from uniform spheroids every day from day 1 to day 7. Diameter, size and invasion into the collagen of the spheroids were measured with ImageJ by evaluating the diameter and area of the spheroids and normalizing it to the initial spheroid at day 1 (mean ± SD, n = 5 for SKMEL23 and TÜMEL173, n≥11 for SKMEL113, n≥23 for TÜMEL62-1, per group). Calcein-AM/PI staining was performed on day 7, and immunofluorescence microphotographs of representative spheroids were taken to determine live or dead cells. White scale bars represent 100 µm. The results were analyzed using Kruskal-Wallis with Dunn’s multiple comparisons test