Figure 6: Digitoxin and MEK inhibitor additively or synergistically impair mitochondrial function and deplete ATP.

(a–c) Flow cytometric analysis of mitotracker deep red fluorescence. (a) Patient-derived xenografts were treated for four days in vivo with MEK inhibitor and/or digitoxin then acutely dissociated and live melanoma cells were analysed by flow cytometry (2–3 independent experiments per melanoma). (b) Mitochondrial membrane potential in human (HLA⁺mCD45⁻) or mouse (HLA⁻mCD45⁺) cells in the blood of NSG mice xenografted with hUCB and treated with digitoxin plus MEK inhibitor for 10 days. (c) Mitochondrial membrane potential of M214 melanoma cells and hiMEL immortalized melanocytes in gelatin sponges transplanted subcutaneously into opposing flanks of mice treated with digitoxin and/or MEK inhibitor for four days. (d–f) Flow cytometric analysis of relative DCFDA fluorescence to measure ROS levels in freshly dissociated melanoma cells (d, 1–3 independent experiments per melanoma), haematopoietic cells (e) or immortalized melanocytes (f) from mice treated with drugs for 4 days in vivo. (g,h) Relative NAD⁺ (g) and ATP (h) levels in 150,000 live melanoma cells sorted from acutely dissociated tumours from 4-day treated mice (each melanoma was tested in 1–2 independent experiments). (i) Relative ATP levels in human haematopoietic cells sorted from the femurs of NSG mice xenografted with hUCB cells and treated with digitoxin plus MEK inhibitor for 4 days. (j) Relative ATP levels in M214 melanoma cells or immortalized melanocytes grown in gelatin sponges transplanted subcutaneously in opposing flanks of NSG mice and treated with digitoxin and/or MEK inhibitor for 4 days. All data represent mean±s.d. In each panel the number of mice per treatment is written on the bars. In a,d,g,h statistical significance was assessed by one-way analysis of variance followed by Dunnett’s multiple comparisons test or the Kruskal–Wallis test followed by Dunn’s multiple comparisons test in cases of unequal variance among treatments. In b,c,e,f,i,j statistical significance was assessed by unpaired two-tailed t-tests. Statistical comparisons of each treatment versus control (NS, not significant; *P<0.05; **P<0.01; ***P<0.001) or the combination compared with MEK inhibitor alone (^#P<0.05; ^##P<0.01; ^###P<0.001) or digitoxin alone (^†P<0.05; ^††P<0.01; ^†††P<0.001).