FIG 1.
Accumulation of Endo G aggregates in the mitochondria induces proteotoxic stress. (A) Localization of GFP or Endo G-GFP (green) was detected by immunofluorescence in cells transfected with the indicated plasmids. Mitochondria (red) were stained with Mitotracker orange dye, whereas nuclei were counterstained with DAPI (blue). Bar, 1 μm. (B) Soluble and insoluble fractions of purified mitochondria treated with sodium carbonate were analyzed by immunoblotting for Endo G, Omi, and cytochrome c. (C) Mitochondrial lysates of cells overexpressing either Endo G-GFP or GFP were used for Western analysis of oxidative phosphorylation (OxoPhos) complex III, the FoF1 ATP synthase α subunit, Omi, VDAC, and Endo G-GFP. (D) Mitochondrial superoxide anion (O2−) production was detected by FACS analysis of live cells transfected with either control GFP or mutant Endo G-GFP as well as of untreated cells or cells treated overnight with 10 μM CCCP or 20 μM rotenone (used as positive controls). (E) Cell viability was measured by an MTS assay at 24, 48, 72, and 96 h after transfection with either the control vector GFP or mutant Endo G-GFP.