FIG. 7.

The ROS inhibitor Tiron inhibits PS-341-mediated apoptosis and gene expression. (A) UMSCC-23 cells were pretreated with or without Tiron for 30 min and then treated with PS-341 for 24 h. Cell viability was determined with a trypan blue exclusion assay. The assay was performed in triplicate, and results represent the average values from three independent experiments. (B) UMSCC-23 cells were pretreated with or without Tiron for 30 min and then treated with PS-341 for 24 h. After treatment, cells were harvested and incubated with the cell-permeable dye H₂DCFDA. The reaction was analyzed by FACS. (C) UMSCC-23 cells were pretreated with or without Tiron for 30 min and then treated with PS-341 for the indicated time periods. The whole-cell proteins were probed with polyclonal antibodies against caspases 9 and 3. For loading control, the membrane was stripped and reprobed with monoclonal antibodies against α-tubulin. (D) UMSCC-23 cells or MEFs were treated with PS-341 with or without Tiron for the indicated time periods, and whole-cell extracts were prepared. Fifty-microgram aliquots were resolved on SDS-8% PAGE and probed with polyclonal antibodies for ATF-4 and CHOP.