Figure 1
Deferasirox causes acute mitochondrial swelling without depolarization. (a) Cell viability in OK cells decreased after 24 hours DFX treatment in a concentration dependent manner (IC50 = 246 µM). Line shows log (inhibitor) versus normalized response variable slope analysis (n = 3). (b) Live confocal imaging in cells loaded with the ΔΨm-dependent dye TMRM showed that DFX (200 µM) causes acute mitochondrial swelling; after 30 minutes mitochondria remained energized (left), but acquired a distinct rounded shape, which was associated with a change in optical density clearly visible in corresponding phase contrast images (right, scale = 10 µm). (c,d) Overlay images of CellLight Mitochondria-GFP BacMam 2.0 (green) and TMRM (red) were used to track the energization state of mitochondria post DFX treatment (scale = 10 µm). The fraction of polarized mitochondria (red line) decreased slowly over time, but even after 3 hours the majority remained energized. Approximately 3,500 mitochondria were sampled at each time point (blue line = the moving average of 40 minutes). (e,f) Swollen mitochondria post DFX treatment (20 minutes) showed an abrupt depolarization in response to FCCP (5 µM, 1 minute), with redistribution of TMRM into the cytosol (n = 4, mean ± standard error of the mean [SEM], ****p < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test, scale = 10 µm). (g) Electron microscopy images showing mitochondrial swelling, decreased matrix density and rupture of mitochondrial membranes in DFX treated cells (arrows, scale = 10 µm in upper images, 1 µm in lower images). (h) Confocal imaging of fixed cells stained with TOM-20 showing no evidence of mitochondrial swelling after 5 hours treatment with the iron chelators deferoxamine (DFA, 200 µM) and deferiprone (DFP, 200 µM, scale = 10 µm). (i) Swelling in isolated mitochondria, measured by a decrease in absorption at 540 nm, occurred after 500 µM Calcium or 5 µM DFX, but not after the addition of the iron chelators deferoxamine (DFA, 2.5 µM) or deferiprone (DFP, 7.5 µM). Concentrations for each drug were chosen to take into account relative iron binding ratios. Values were normalized to the first measurement after drug addition (n = 4, mean ± SEM).
