Figure 6.
Effect of Mfn2 blockade on MCI mitochondrial morphology and function. (A) Quantification of immunoreactive bands for Mfn2 normalized to β-actin in MCI cybrid cell lysate of the indicated group with siRNA-control and siRNA-Mfn2 transfection. Representative immunoblots are shown in the lower panel. Data are expressed as fold increase relative to siRNA-control transfected MCI cybrid cells. (B) MCI cybrid cells were transfected with siRNA-control or siRNA-Mfn2. After 24 h, cells were incubated with Mitotracker Red to analyze mitochondrial morphology using confocal microscopy (Scale bar = 5 µm). siRNA-Mfn2 transfected MCI cybrids had tubular mitochondria, whereas siRNA-control transfected cells retained elongated mitochondrial morphology. Mitochondrial average density (C), length (D), and CcO activity (E), and mitochondrial membrane potential (TMRM) (F) were restored in siRNA-Mfn2 transfected cells compared to siRNA- transfected control cells. ROS production measured by Mitosox (G) and EPR values (H, I) were suppressed in siRNA-Mfn2 transfected cells compared to siRNA-transfected control cells. (H) Quantification of EPR values in the indicated groups of cells. (I) Representative EPR spectra. (J,K) Representative images for Mitotracker and Mitosox staining are shown (Scale bar = 10 µm). N = 5–7 cell lines/group.