Fig. 2. Effects of mitoTRAM-34 and rev-mitoTRAM on mitochondrial physiology.
a Representative Western Blot showing the mitochondrial localization of KCa3.1 in B16F10 cells. f1: whole-cell extract; f2: membrane-enriched fraction; f3: mitochondria-enriched fraction; m1 and m2: Percoll-purified mitochondrial fractions (see “Materials/subjects and methods”). Plasma membrane marker PMCA and mitochondrial membrane markers VDAC-1 and TOM-20 are also shown. b, c Images showing mitochondrial membrane potential changes (b) and superoxide production (c) in B16F10 cells, as visualized by changes in TMRM (b) or mitoSOX (c) fluorescence upon addition of mitoTRAM-34 (mitoT.), rev-mitoTRAM (rev-m.) or TRAM-34 at the indicated time points. FCCP (b) and Antimycin A (c) were used as controls (not shown). The quantification of the fluorescence signal is shown on the right. Fluorescence is expressed as percentage of the initial intensity (mean + SEM; Two-Way Anova with Dunnett’s multiple comparison test. N = 4. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control). Scale bar is 100 µm. d Representative images (left) and analysis (right) of mitochondrial ultrastructure assessed by transmission electron microscopy (TEM) after treatment of B16F10 cells with 1.5 µM mitoTRAM (mitoT.) or 7.5 µM rev-mitoTRAM (rev-m.) for 30 min. The mitochondrial circularity and number of cristae/area were calculated with ImageJ. For each replicate, mitochondria from at least 5 cells were analyzed (mean + SEM, One-Way Anova with Dunnett’s posttest. N = 3. ****p < 0.0001 compared to control). Scale bar is 1 µm. e As in (d), but cells were treated for 24 h with sublethal doses of mitoTRAM-34 (0.5 µM) and rev-mitoTRAM (5 µM). f Representative confocal images of B16F10 cells treated as in (e) showing a fragmented mitochondrial network after 24 h of treatment with mitoTRAM-34 (mitoT.) or rev-mitoTRAM (rev-m.) compared to control cells. Cells were stained for mitochondrial marker TOM-20 (magenta) and DAPI (cyan). Scale bar is 5 µm.