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Fig. 7. Toxoplasma infection fails to block the activity and activation of caspase 3 in p65–/– fibroblasts. The effect of T. gondii infection (20 hours) on the activity of caspase 3 following treatment with STS (A,B) or TNF{alpha} (A,C) for 8 hours was examined over a broad concentration range. Caspase 3 activity was detected using the intrinsic substrate PARP (A, PARP). Unlike wild-type fibroblasts, infected p65–/– fibroblasts exhibit an identical level of the cleaved 89 kDa form as uninfected cells under both STS (top) and TNF{alpha} (middle) treatment. The complete cleavage of PARP in CX/TNF{alpha} samples reflects their hypersensitivity to TNF{alpha}-mediated apoptosis. Identical results were obtained in the absence of CX (data not shown). The cleavage of PARP correlates with the activation of caspase 3, which is not inhibited in Toxoplasma-infected p65–/– fibroblasts (A, bottom). Immunoblots in the middle and bottom panels were performed on the same source material. Activity of caspase 3 measured using the fluorescent substrate (DEVD-MCA) cleavage assay supports the immunoblot data, indicating that no protection is afforded by T. gondii infection (white bars) compared with uninfected p65–/– cells (gray bars) across a broad concentration range following STS treatment (B). Similarly, TNF{alpha} treatment triggers a dramatic activation of caspase 3 (notice scale for relative fluorescence) in both infected (white bars) and uninfected (gray bars) cells (C). This is consistent with the complete cleavage of PARP observed by immunoblot analysis. The apparent partial protection observed with infected cells at lower concentrations of TNF{alpha} might indicate that there might be an alternate mechanism of protection independent of NF-{kappa}B function. Alternatively, death might be more rapid in infected cells with highly activated caspase 3, resulting in the loss and lysis of a significant population prior to harvesting. In the case of induction of apoptosis by TNF{alpha}, the addition of cycloheximide is noted by a plus (+) sign. Error bars represent standard deviations using triplicate samples.