Figure 4: Climacostol activates mitochondrial-dependent apoptosis in melanoma cells. | Scientific Reports

Figure 4: Climacostol activates mitochondrial-dependent apoptosis in melanoma cells.

From: Climacostol reduces tumour progression in a mouse model of melanoma via the p53-dependent intrinsic apoptotic programme

Figure 4

B16-F10 cells were cultured in the presence of 30 μg/ml climacostol or vehicle (control) for 24 h. (a) Evaluation by flow cytometry of mitochondrial potential, using TMRM staining. The percentage number of cells with intact mitochondrial potential (TMRM fluorescence in top gate) and percentage number of cells with reduced mitochondrial potential (depolarised cells; TMRM fluorescence in bottom gate). FSC: forward scatter. Images and data are representative of 3 independent experiments. (b) Percentage of depolarised cells for experiments shown in (a). *p < 0.0001 vs control. (c,d) Confocal microscopy for co-localization of Bax and Cytochrome c with mitochondria. Cells were stained for Bax or Cytochrome c (red) and the fluorescent mitochondrial dye MitoTracker Green. DAPI (blue) was used for nuclei detection. The images are representative of 3 independent experiments. Scale bar: 20 μm. Panels on the right represent enlarged image details marked by the white arrows. (e) Immunofluorescence imaging of cleaved Caspase 9 (punctate red pattern). DAPI (blue) and phalloidin (green) were used for nuclei and cytoskeleton detection, respectively. The images are representative of 3 independent experiments. Upper panels: 100 μm scale bar; lower panels: 50 μm scale bar.

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