Figure 1: Shikonin accumulates in mitochondria and causes a breakdown of the mitochondrial membrane potential. (a) 2D excitation (200–600 nm) versus emission (300–700 nm) fluorescence spectrum of 50 μM shikonin in aqueous buffer. (b) Real-time kinetics and quantification of cellular shikonin uptake by flow cytometry. The inherent fluorescence of intracellular shikonin was measured at 640 nm excitation with a 730/45 nm bandpass filter. A dose-dependent increase of the cellular shikonin fluorescence was observed after treatment with increasing concentrations (0.15, 0.3 and 0.6 μM) of shikonin. After 20 min of incubation with 0.6 μM shikonin and subsequent washing of the cells, shikonin’s fluorescence was still detectable in cells, indicating persistent intracellular accumulation of shikonin. (c) Shikonin localizes to mitochondria. U937 or SK-BR-3 cells were stained with MitoTracker Green and subsequently treated with 25 μM shikonin. Cells were then examined by confocal microscopy at an excitation wavelength of 488nm and 561 nm and emission at 500–549 nm and 680–780 nm for MitoTracker Green and shikonin, respectively. (d) Breakdown of the mitochondrial membrane potential. U937 cells were stained with JC-1, which has a strong red fluorescence in healthy mitochondria. Shikonin induced a dose-dependent decrease of the red JC-1 fluorescence after 6 h of treatment with increasing concentrations of shikonin (0.3, 0.6, and 1.2 μM), indicating a breakdown of the mitochondrial membrane potential.