FIGURE 2: Infection of host cells with T. gondii diminishes in vitro and in vivo activity of caspase 3/7 triggered by exogenous cytochrome c. (A, B) Jurkat T cells were infected with increasing amounts of T. gondii (multiplicity of infection (MOI) 10:1 to 30:1; cross-hatched bars or as indicated) or were left non-infected (black bars or as indicated).
(A) After 24 hours of infection, cytosolic extracts were isolated and the caspase cascade was activated using cytochrome c/dATP. Caspase 3/7 activity was determined by fluorimetric measurement of DEVD-AMC cleavage over time. Results represent means ± S.E.M. (n=3).
(B) Alternatively, the cytosolic extracts from Jurkat cells and total extract from the same numbers of parasites as having been used for infection were separated by SDS-PAGE and analyzed by immunoblotting using antibodies which recognize T. gondii SAG1 or host cell actin. Bound antibodies were visualized using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection. Results are representative for two independent experiments.
(C) Jurkat cells infected with T. gondii for 1 hour (MOI 30:1; cross-hatched bars) and non-infected control cells (open bar and black bars) were electroporated in the presence of cytochrome c (cytc) or bovine serum albumin (ctrl) or were left non-treated. After incubation of the cells and subsequent cell lysis, caspase activity was determined by fluorimetric measurement of the cleavage of the caspase 3/7 substrate DEVD-AMC. Data represent the increase of cleavage over time; bars indicate means ± S.E.M. (n=6). Significant differences were identified by Student’s t-test (*** P < 0.001).
(D) After infection and/or electroporation of cells as described above (C), cells were partitioned into a digitonin-soluble fraction comprising the host cell cytosol and a digitonin-insoluble fraction including mitochondrial proteins. Proteins were resolved by SDS-PAGE and after transfer to nitrocellulose, were immunolabelled using antibodies recognizing cytochrome c (cyt c), cytochrome c-oxidase subunit IV (COX) or actin. Immune complexes were visualized using peroxidase-conjugated secondary antibodies and enhanced chemiluminescence detection. Similar results were obtained in two independent experiments.