Figure 7.

Effect of OGC and DIC inhibition on ETC proteins and mitochondrial morphology of RPE. (A) RPE cells were treated with and without αB cry peptide (75 µg/mL) for 24 h and total cellular protein was analyzed by Western blot under non-reducing conditions for ETC proteins using a commercial ETC complex cocktail antibody. mGSH transport inhibition resulted in decreased expression of complex proteins I and II and αB cry peptide treatment upregulated these protein subunits. Please note that the bands for complexes I and IV appeared overlapping each other making the interpretation of changes in complex I expression difficult. Complexes I and IV and complexes III and V are separated by dotted white lines in the figures for easy visualization. (B) Protein expression of mtTFA was measured by Western blot analysis. PS or BM treatment reduced mTFA expression, whereas co-treatment with αB cry peptide upregulated mTFA expression (C). RPE cells were grown on 4-well chamber slides and without αB cry peptide (75 µg/mL) for 24 h. At the end of the treatment, cells were incubated with MitoTracker Red (100 nM) for 10 min. The samples were washed, fixed in 4% PFA and viewed under LSM 770 confocal microscope. Mitotracker (Red) and DAPI (Blue) staining of RPE cells with and without treatment. In RPE cells treated with PS (5 mM) or BM (5 mM), mitochondria appeared fragmented while in the αB cry peptide group mitochondria are perinuclear, similar to that seen in control cells. These data show that inhibition of either OGC or DIC results in an altered mitochondrial arrangement. Scale bar = 5 µm.