Figure 5: Digitoxin and MEK inhibitor synergistically dysregulate mitochondrial Ca²⁺ levels.

(a) Live-cell laser scanning confocal imaging of fluo-3 fluorescence in A375 melanoma cells to measure cytoplasmic Ca²⁺. (b) Representative images of rhod-2 fluorescence in the mitochondria of cultured A375 cells before or 700 s after addition of 25 nM digoxin and 10 nM MEK inhibitor. Mitochondrial rhod-2 staining was confirmed by MitoTracker co-localization (Supplementary Fig. 7g). (c–k) Live-cell laser scanning confocal microscopy of rhod-2 fluorescence in the mitochondria of cultured A375 (c), M481 (d), M491 (e) or M214 (f) melanoma cells or normal human haematopoietic cells (g), two lines of primary human melanocytes (h,i) or cultured melanoma cells expressing vector (tRFP control) or NHE1 (j,k) to measure mitochondrial Ca²⁺ levels. For each treatment 6–12 mitochondrial regions from each of 3–6 cells were continuously imaged for 30–37 min. The first arrow indicates the addition of 50 nM digitoxin and/or 10 nM MEK inhibitor. The second arrow indicates the addition of 10 μM FCCP to uncouple and dissipate mitochondrial membrane potential. Statistical significance was assessed by a two-way ANOVA followed by Dunnett’s multiple comparisons test at 15 min (c) or 25 min (d–k). (l,m) Activated caspase-3⁺ cells in sections of M214 melanomas (l) and flow cytometric analysis of intracellular pH (m) in dissociated M214 xenografts from mice treated for four days with digitoxin plus MEK inhibitor and/or BAPTA-AM (a calcium chelator). Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple comparison’s test. All data represent mean±s.d. Each treatment compared with 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).