FIGURE 6.

Celstrol-induced ROS is involved in CHOP induction leading to DR5 up-regulation. A, MDA-MB-231 cells were treated with 3 μmol/liter celastrol with or without 10 mmol/liter NAC. Twelve hours later, intracellular ROS levels were measured by flow cytometry using CM-H₂DCFDA, as described under “Experimental Procedures.” B, cells (5 × 10⁵ cells) were pretreated with various concentrations of NAC or GSH for 1 h and then treated with 3 μmol/liter celastrol for 24 h. Whole cell extracts were prepared and analyzed by Western blotting using DR5 antibody. C, cells were treated with either NAC or GSH for 1 h and exposed to 3 μmol/liter celastrol for 12 h, and then whole cell extracts were subjected to Western blotting for CHOP. The same blot was stripped and reprobed with β-actin antibody to verify equal protein loading. D, MDA-MB-231 cells were pretreated with NAC for 1 h and then treated with celastrol for 6 h. Next cells were washed with PBS to remove celastrol and treated with TRAIL for 24 h. Cell death was determined by the Live/Dead Assay.