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. 2011 Jun;163(3):567–585. doi: 10.1111/j.1476-5381.2011.01233.x

Figure 2.

Figure 2

2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) induces caspase-independent apoptotic cell death. (A) NAD(P)H quinone oxidoreductase 1 (NQO1)+-MDA-MB-231 cells were treated with 10 µM RH1 for the indicated times. Whole cell lysates were prepared and evaluated by Western blotting for expression of active caspases-8, -9 and -3; polyADP ribose polymerase (PARP); and β-actin. The relative density value of each band is shown below the Western blot. The data are representative of a typical experiment that was conducted three times (mean values, P < 0.05). (B) NQO1+-MDA-MB-231 cells were treated with 10 µM RH1 for 36 h in the presence or absence of 30 µM z-VAD-fmk. Whole cell extracts were directly examined by Western blotting using anti-active caspase-8, -active caspase-9, -active caspase-3, -PARP and -β-actin antibodies (left panel). The relative density value of each band is shown below the Western blot. The data are from a typical experiment that was conducted three times with similar results (mean values, P < 0.05). Cells were stained with propidium iodide (PI) and apoptotic cell numbers were measured by flow cytometry. Results of three independent experiments are expressed as means ± SEM. *Significant difference between control and RH1-treated cells at P < 0.05. **Significant difference between RH1-treated cells and z-VAD-fmk + RH1-treated cells at P < 0.05. (C) NQO1+-MDA-MB-231 cells were treated with 10 µM RH1 for 12 h in the presence or absence of 30 µM z-VAD-fmk. Cells were stained with Annexin V/PI and apoptotic changes were assessed by flow cytometry. Representative flow cytometric dot plots are shown: lower left quadrant, viable cells; lower right, early apoptotic cells; upper right, late apoptotic cells; upper left, necrotic cells. Apoptotic changes were quantitated as described below. Results from three independent experiments are expressed as means ± SEM. *Significant difference between control and RH1-treated cells; P < 0.05. **Significant difference between RH1-treated cells and z-VAD-fmk + RH1-treated cells; P < 0.05.