Figure 5.

2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone (RH1) activates mitogen-activated protein kinases (MAPKs) in NAD(P)H quinone oxidoreductase 1 (NQO1)⁺-MDA-MB-231 cells. (A) NQO1⁺-MDA-MB-231 cells were treated with 10 µM RH1 for the times indicated. Whole cell lysates were subjected to Western blot analysis using anti-phospho-c-Jun N-terminal kinase (JNK)1/2, -JNK1/2, -phospho-p38 MAPK, -p38 MAPK, -phospho-extracellular signal-regulated kinase (ERK)1/2, -ERK1/2 and -β-actin antibodies. 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). (B) NQO1⁺-MDA-MB-231 cells were treated with 10 µM RH1 for 36 h in the presence or absence of 30 µM SP600125, SB203580 or PD98059. Cells were stained with PI and apoptotic cell numbers were measured by flow cytometry. 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- and SP600125 + RH1-treated cells; P < 0.05. (C) NQO1⁺-MDA-MB-231 cells were treated with 10 µM RH1 for 6 h in the presence or absence of 30 µM SP600125, SB203580 or PD98059. Cells were loaded with 30 nM of 3,3′-dihexyloxacarbocyanine iodide [DiOC₆(3)] during the last 30 min of RH1 treatment. After removal of medium, the concentration of retained DiOC₆(3) was measured by flow cytometry. The 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- and SP600125 + RH1-treated cells; P < 0.05. (D) NQO1⁺-MDA-MB-231 cells were treated with 10 µM RH1 for 36 h in the presence or absence of siRNAs targeting JNK1 or JNK2. Cells were stained with PI and apoptotic cell numbers were analysed by flow cytometry. Results from three independent experiments are expressed as means ± SEM. *Significant difference between control and RH1-treated cells; P < 0.05. **Significant difference between si-cont RNA- and si-JNK1 RNA-transfected cells in response to RH1 treatment; P < 0.05. ***Significant difference between si-cont RNA- and si-JNK2 RNA-transfected cells after RH1 treatment; P < 0.05. (E) NQO1⁺-MDA-MB-231 cells were treated with 10 µM RH1 for 6 h in the presence or absence of siRNA targeting JNK1 or JNK2. Cells were loaded with 30 nM of DiOC₆(3) during the last 30 min of RH1 treatment. After removal of medium, the concentration of retained DiOC₆(3) was measured by flow cytometry. Results from three independent experiments are expressed as means ± SEM. *Significant difference between control and RH1-treated cells; P < 0.05. **Significant difference between si-cont RNA- and si-JNK1 RNA-transfected cells after RH1 treatment; P < 0.05. ***Significant difference between si-cont RNA- and si-JNK2 RNA-transfected cells after RH1 treatment; P < 0.05.