Fig. 3. Inhibition of caspase 3 activity in T.-gondii-infected cells. The effect of T. gondii infection (20 hours) on the activity of caspase 3 following treatment with STS (A,C) or TNF (B,D) for 8 hours was examined over a broad concentration range. Caspase 3 activity was detected using the intrinsic substrates 2-spectrin (A,B, Spectrin) or PARP (A,B, PARP). Caspase 3 activity can be detected in immunoblot analysis by the generation of the specific 120 kDa spectrin fragment (A,B, Spectrin, arrowhead) and the 89 kDa PARP fragment (A,B, PARP, arrowhead). Uninfected cells exhibit these characteristic bands with increasing concentrations of the apoptogenic trigger. By contrast, T.-gondii-infected cells exhibit a significant inhibition of caspase 3 activity. Low levels of activity in the infected sample are attributable to uninfected cells or, in the case of STS, probable toxicity towards the parasite. Caspase 3 activity was also measured in STS (C) and TNF (D) treated cells using the direct cleavage of a fluorescent substrate (DEVD-MCA). Activity was determined based on the fluorescence caused by the release of MCA. Based on the fluorometric analysis, infected cells (white bars) exhibit considerably lower levels of activity than uninfected cells (gray bars) across a broad range of concentrations of STS (C) and TNF (D). In the case of induction of apoptosis by TNF, the addition of cycloheximide is noted by a plus (+) sign. Error bars represent standard deviations using triplicate samples.

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