Fig. 6

Copper uptake in BRAF WT melanoma cells by CuET and its combination with trametinib. A Copper uptake assay measuring Cu²⁺ concentrations in lysates and subcellular subfractions of BRAF WT melanoma cell lines (SKMEL23 and SKMEL113) after treatment with trametinib (10 nM), CuET (125 nM) and the combination for 6 hours. Untreated melanoma cells were used as a control (n = 3, mean ± SD). B Confocal immunofluorescence analysis for Cu(I) via CooperGreen™ staining in BRAF WT melanoma cells (SKMEL113) after treatment with CuET (125 nM) in the presence or absence of (+/-) 10 nM MEK inhibitor trametinib for 6 hours. Nuclei were stained with DAPI (blue). Scale bars represent 25 μm. Confocal immunofluorescence analysis and fluorescence intensities of the copper probe were recorded as arbitrary fluorescence units (n ≥ 90 cells/ group were analyzed). C Spectrophotometric copper uptake assay measuring intracellular Cu²⁺ concentrations in melanoma cell lines (SKMEL23 and SKMEL113) transfected with siRNA directed against ATOX1 or control siRNA followed by treatment with the combination of trametinib (10 nM) plus CuET (125 nM) for 6 hours (n=3, means ± SD). D Cell viability assay of BRAF WT melanoma cell lines (SKMEL23 and SKMEL113) transfected with control siRNA or siRNA directed against ATOX1 following treatment with the MEK inhibitor trametinib (10 nM) plus increasing concentrations of CuET (up to 500 nM) for 72 hours. Treatment started 24 hours post transfection (n = 3; mean ± SD). One-way ANOVA followed by Tukey’s multiple comparisons test was applied in (A), (B) and (C) with * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001, ns (not significant)