Activation of caspase 3 in CEM/ADR5000 cells after FeNG treatment.

<p>(A) Effect of FeNG and caspase inhibitors on the activity of caspase-3 of CEM/ADR5000 cells. Cells were treated with either vehicle (medium) control or FeNG (10⁻⁴ M) for 12 h, 24 h, 48 h, 72 h or caspases inhibitors; Ac-DEVD-cho (caspase 3 specific inhibitor) and z-VAD-fmk (pan caspase inhibitor) alone (50 µM) or in combination with FeNG for 72 h. After completion of these treatments, cells were harvested and cell lysates were prepared. The enzymatic activity of cell lysates towards tetrapeptide chromogenic substrates Ac-DEVD-AMC was determined. Caspase activities are expressed as fold change of control and presented as mean±SD of three independent experiments. Differences between untreated control and FeNG treated cells are significant ***P<0.001, by unpaired Student's t test. (B) Effects of caspase inhibitor on FeNG induced cell death of CEM/ADR5000 cells. Cells were either left untreated or treated with FeNG (10⁻³ M or10⁻⁴ M) or Ac-DEVD-cho (50 µM) and z-VAD-fmk (50 µM) alone or in combination with FeNG for 72 h and cell death was monitored by MTT assay. Value represents the mean ± SD of three independent experiments with four replicates in each. Significant difference at *P<0.05, **P<0.01, respectively, from only FeNG treated cells. (C) Effect of FeNG on the cleavage patterns of PARP in CEM/ADR5000 cells. Cells were grown at standard culture conditions as mentioned in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0011253#s4" target="_blank">Materials and methods</a>, and treated with FeNG (10⁻⁴ M) for 24 h to 72 h, and cell lysates were prepared. Immunoblot analysis was performed to identify the full (116 kDa) and cleaved (89 kDa) PARP using specific primary antibodies. Loading was checked by immunoblotting of β -actin. Bands were visualized by Lumi glow detection system. Data shown are representative of three independent experiments. (D) Densitometric quantitation of cleaved (89 kDa) frgments of PARP in the cytoplasm. Immunoreactive bands were quantitated and expressed as the ratio of each band density to corresponding loading control (β actin) band density and values were represented after normalization to untreated control.