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. 2006 Dec 27;81(6):2792–2804. doi: 10.1128/JVI.01760-06

FIG. 3.

FIG. 3.

Functional Fas and PKR are required for rM51R-M virus-induced apoptosis. (A) L929 cells stably expressing an empty vector control plasmid (EV) or dominant-negative mutants of Fas (ΔFas) or PKR (ΔPKR) were infected with rM51R-M virus. The cumulative percentage of cells entering apoptosis was determined using time-lapse video microscopy and is plotted as a function of time postinfection. The data represent the averages ± SEM of three experiments. (B) L929-EV, L929-ΔFas, and L929-ΔPKR cells were infected with rWT virus. Cells entering apoptosis were monitored using time-lapse video microscopy as described above (A). The data represent the averages ± SEM of three experiments. (C) Fluorometric caspase-8 assays were performed using L929-EV, L929-ΔFas, and L929-ΔPKR cells that were mock infected or infected with rM51R-M virus. At the indicated times postinfection, cells were lysed, and caspase-8 activity was measured with a fluorogenic substrate. Caspase-8 activity is expressed in arbitrary fluorescence units per mg of total protein. The data represent the average ± SEM of three experiments. (D) Caspase-3 activity was measured via flow cytometry using PE-conjugated antibodies against active caspase-3 in L929-EV, L929-ΔFas, and L929-ΔPKR cells that were mock infected or infected with rM51R-M virus. Data are expressed as the geometric mean fluorescence intensity (MFI) normalized to the maximum level observed in EV cells. The data represent averages ± SEM of three experiments. (E) Levels of PKR phosphorylation at threonine 446 were measured by flow cytometry using a phosphospecific antibody in L929, L929-EV, L929-ΔFas, and L929-ΔPKR cells that were mock infected or infected with rM51R-M virus. Data are expressed as the geometric mean fluorescence intensity (gMFI) (average of two independent experiments). (F) Apoptosis in the presence and absence of 2-AP was measured via flow cytometry by staining with annexin V and 7-AAD. Data are presented as percentages of annexin V-positive cells and represent averages ± SEM of three experiments.