NAC blocks GO-203–induced decreases in GSH and cell death. (A-C) U266 and RPMI8226 cells were left untreated, and treated with 5μM GO-203 or 5μM CP-2 each day for 3 days. The GO-203–treated cells were also incubated in the presence of 5mM NAC for the last 2 days. (A) The U266 (left) and RPMI8226 (right) cells were analyzed for GSH levels. The results are expressed as relative GSH levels (mean ± SD of 3 determinations) compared with that obtained with the control. (B-C) The U266 (B left) and RPMI8226 (C left) cells were incubated with PI and annexin V, and analyzed by flow cytometry. The results are expressed as the percentage of cells with late apoptosis/necrosis (mean ± SD of 3 determinations; B and C right). (D) U266 (left) and RPMI8226 cells (right) were left untreated, and treated with 5μM GO-203 each day for 3 days. The GO-203–treated cells were also incubated in the presence of 5mM GSH for 3 days. The cells were then incubated with PI and annexin V, and analyzed by flow cytometry. The results are expressed as the percentage of cells with late apoptosis/necrosis (mean ± SD of 3 determinations). (E) RPMI8226 cells were left untransfected, and transfected with control (CsiRNA) or TIGAR siRNA pools for 72 hours. TIGAR and β-actin mRNA levels were determined by RT-PCR (left). Analysis of the intensity of the signals by densitometric scanning demonstrated values of 0.98 for CsiRNA-treated cells and 0.29 for TIGER siRNA-treated cells relative to that obtained for nontransfected cells (assigned a value of 1.0). The transfected cells were left untreated, and treated with 5μM GO-203 for 24 hours. Viable cell number (mean ± SD of 3 determinations) was determined by trypan blue exclusion (right). (F) Schematic representation of the proposed effects of inhibiting MUC1-C on the disruption of redox balance in multiple myeloma cells.