Figure 5.

RA183 triggers unresolved UPS stress and apoptosis. (A) After treatment of ES2 cells with bortezomib or RA183 (1 μM) for the indicated times, mRNA was extracted and CHOP-10 mRNA levels were assessed by quantitative real-time polymerase chain reaction (PCR) and normalized to GAPDH expression. Results are expressed as fold change over control-treated cells and represent the average and SD of three independent experiments. (B) Same as in (A), except BIP mRNA levels were assessed. (C) Same as in (A), except XBP1 spliced mRNA levels were assessed. (D) Western blot analysis for ATF-4 and actin in OVCAR3 (left) and ES2 (right) cells either untreated (control) or treated with 1 μM RA183 for 6 h. (E) After treatment of ES2 cells with 1 μM RA183 for the indicated times, ES2 cell lysate was subjected to Western blot analysis and probed with antibody to PARP (top) and Bax (middle) or actin (bottom). (F) OAW42 cells were treated with RA183 (empty) or vehicle control (filled) for 12 h, respectively. After treatment, the cells were harvested and incubated with the cell-permeable dye H₂DCFDA. Release of dichlorofluorescein was analyzed by flow cytometry as a measure of reactive oxygen species (ROS). H₂O₂ was used as a positive control (not shown). (G, H) SKOV3 and TOV21G cells were treated with 1 μM b-AP15 or RA183 at 0.5 or 0.25 μM for 12 h and analyzed by flow cytometry after staining with fluorescein-labeled annexin-V.