FIG. 2.
PV-induced mitochondrial damage in neuronal cells. (A) Representative flow cytometric histograms after DiOC6 staining of mock-infected (gray area) and PV-infected (6 h p.i.; white area) IMR5 cells. The percentages of apoptotic cells corresponding to a reduced fluorescence intensity (ΔψmLow) for each of the two experimental conditions are indicated. (B) Flow cytometric analysis of mitochondrial-permeability transition in PV-infected IMR5 cells. Mock-infected, PV-infected, and STS-treated IMR5 cells were analyzed at the indicated times p.i. by flow cytometry after DiOC6 staining, and the percentages of ΔψmLow cells are shown in white, light gray, and dark gray, respectively. The graph reports the mean percentages of ΔψmLow cells obtained from three independent experiments. Error bars represent the standard errors of the means. *, P < 0.05 by Student's t test comparing PV-infected and STS-treated IMR5 cells to mock-infected IMR5 cells. (C) Time course of cytochrome c redistribution in PV-infected IMR5 cells. At the indicated times p.i., cells were collected and subjected to subcellular fractionation. The cytochrome c (Cyt c) was detected in the cytosolic fraction by Western blotting analysis with an anti-cytochrome c antibody. Mock-infected IMR5 cells and cells treated with STS for 8 h were used as negative and positive controls, respectively. Levels of actin were used to control for protein loading. (D) Cytochrome c redistribution in PV-infected IMR5 cells. Mock-infected and PV-infected IMR5 (6 h p.i.) cells were stained by immunofluorescence with a specific monoclonal antibody against cytochrome c and a secondary, fluorescein-conjugated antibody. See the text for details. (E) Caspase-9 activation in PV-infected IMR5 cells. Caspase-9 activation was determined in mock- and PV-infected IMR5 cells (6 h and 8 h p.i.) by flow cytometry, using a fluorescein-labeled inhibitor (FAM-LEHD-fmk) that binds specifically to active caspase-9, as described in Materials and Methods. A histogram representative of two independent experiments is shown. The percentages of cells positive for activated caspase-9 are indicated.