Figure 3. Abnormal mitochondria accumulate in cells overexpressing mutant p150^glued.

(A) HeLa cells transfected with GFP-empty vector, GFP-tagged wild-type or mutant (G59S or G71R) p150^glued were incubated with MitoTracker Deep Red (100 nM) for 15 min and analyzed using confocal microscopy. Insets show higher magnification of the boxed areas. Bars, 10 μm. (B, C) Twenty-four hours after transfection, GFP-positive HeLa cells were sorted using flow cytometry and analyzed by immunoblotting with antibodies against TOM20 and actin (B). Densitometry analysis of TOM20 levels relative to actin was performed in three independent experiments (C). (D, E) Twenty-four hours after transfection, HeLa cells were incubated with Mitotracker-Red CMXRos (25 nM) for 15 min, and intracellular fluorescence intensity was measured by flow cytometry. The histograms of MitoTracker-Red CMXRos fluorescence in GFP-positive cells (D) and the percentages of GFP-positive cells with reduced mitochondrial potentials (E) are shown. The error bar indicates each standard deviation. Statistics are from three independent experiments: *, p<0.05; **, p<0.01; ***, p<0.001.