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Figure 1. A) Evidence of in situ apoptotic DNA fragmentation in liver during P. yoelii infection, as detected by TUNEL assay. Control (uninfected) liver (a): there is no typical staining for apoptotic DNA fragmentation. Infected liver (b): profuse staining as indicated by black dots, suggesting DNA fragmentation. Arrows indicate TUNEL-positive cells. DNase treated control liver (positive control). B) Activation of caspase-3 like proteases in liver of P. yoelii infected mice. Activity of caspase-3 in the cytosolic fraction (50 µg protein) was measured at different timepoints. Caspase-3 activity was expressed as change of optical density at 405 nM due to release of pNA. To check specificity of caspase-3 assay, Ac-DEVD-CHO, a specific inhibitor was added. Data are mean ± SE (n=6). C) Immunocytochemical detection of activated caspase-3 in isolated hepatocyte. Hepatocytes from control (uninfected; a) and infected (b) mice were used for immunocytochemistry using caspase-3 Ab and activation of caspase-3 is indicated by black spots (arrow). D) RT-PCR for Bcl-2, Bax, and Fas. Liver RNA (1 µg) from infected or uninfected (control) mice was used for RT-PCR amplification. Amplified products were checked in 12% polyacrylamide gel. GAPDH was used as internal (positive) control. E) Densitometric analysis of Bcl-2, Bax, and Fas expression (percent relative to control, where control was considered as 100% expression) in liver of infected mice. Data were calculated from 3 separate experiments and are mean ± SE (***P<0.001 vs. control). F) Measurement of caspase-8 activity. Pure caspase-8 was used as positive control. Inhibition of caspase-8 activity by caspase-8 specific inhibitor (IETD-CHO) indicated specific assay for caspase-8. Data are mean ± SE.
