Involvement of caspase-dependent and caspase-independent modes of cell death.

<p>(A) Caspase activation. Activity of caspase-3, -8, -9 was measured in control and F2 (18.6 µg/ml) treated U937 cells (2.5×10⁵/ml) using colorimetric tetrapeptide substrates. Results are expressed as mean ± SEM from three independent experiments. (B) Cleavage of PARP by F2. Cell lysates were prepared and subjected to western blot analysis to check the cleavage of PARP. Data shown are from one of the three experiments. (C) Effect of pan caspase inhibitor on F2 induced cytotoxicity. Cells (2.5×10⁴/100 µl of RPMI 1640 medium/well) were pre-incubated with pan caspase inhibitor Z-VAD-FMK (20 µM; 4 h) followed by treatment with F2 (0–100 µg/ml; 48 h). Cell viability was evaluated by MTT assay. Histograms represent percentage cell viability (Mean ± SEM) obtained at the IC₅₀ concentration of F2 (18.6 µg/ml) and has been derived from at least three experiments in duplicate. (D) Effect of pan caspase inhibitor on F2 induced apoptosis. Cells were treated with F2 (18.6 µg/ml; 48 h) with or without pre-incubation using Z-VAD-FMK (20 µM; 4 h). They were co-stained with Annexin V-FITC and PI followed by analysis using flow cytometry. Histograms represent percentage of apoptotic cells and have been derived from at least three experiments (***p<0.001 compared to control cells). (E) Translocation of AIF. Cytoplasmic and nuclear fractions were prepared from control and F2 treated U937 cells using nuclear/cytosol fractionation kit and translocation of AIF was analyzed by Western blotting. Analysis was confirmed with three different sets of extracts. Laminin served as a loading control.