Figure 6.

DBA induces generation of ROS and DBA-induced up-regulation of DR5 and DR4 was mediated by ROS. (A) HCT116 (1×10⁶ cells) cells were labeled with DCF-DA, treated with indicated concentration of DBA for 1 h and examined for ROS production by flow cytometer (Left panel). HCT116 cells (1×10⁶ cells) were pretreated with various concentrations of NAC for 1 h and then the cells were treated with 15 µM DBA for 24 h. Whole-cell extracts were prepared and analyzed by Western blotting (Right panel). (B) NAC reverses the DBA-induced inhibition of antiapoptotic proteins. HCT116 cells were pretreated with NAC for 1 h and then treated with 15 µM DBA for 24 h. Whole-cell extracts were prepared and subjected to Western blotting. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (C) NAC reverses cell death induced by combination of DBA and TRAIL. HCT116 cells were pretreated with NAC (10 mM) for 1 h and then treated with 15 µM DBA for 12 h. After washing with PBS cells were treated with TRAIL (25 ng/mL) for 24 h. Cell death was determined by the Live/Dead assay. Percent dead cells are mentioned below the photo. (D) NAC inhibited caspase activation and PARP cleavage induced by combination of TRAIL and DBA. HCT116 cells were treated with NAC, DBA and TRAIL as indicated above. Whole-cell extracts were prepared and analyzed by Western blotting using the relevant antibodies. β-actin was used as a loading control.