Induction of ROS and Noxa was involved in myeloma cell death. (A) PRIMA-1^Met induced ROS production. XG6 (TP53^wt), OPM2 (TP53^R175H), and JJN3 (TP53^neg) cells (0.5 × 10⁶/mL) were incubated overnight with 20 μM (JJN3) or 30 μM (XG6, OPM2) PRIMA-1^Met in the presence or absence of 5 mM NAC. CellROX reagent (5 μM) was added to the cell culture for the last 30 minutes, at 37°C. Cells were washed in phosphate-buffered saline, and fluorescence was analyzed on FACSCalibur. (B) ROS scavengers NAC and GSH-MEE inhibited PRIMA-1^Met-induced cell death. Cells were treated with PRIMA-1^Met for 2 days in the presence or absence of NAC or GSH-MEE (5 mM). Cell death was assessed using Apo2.7 staining. The data represent the mean ± SEM of at least 3 experiments. ***P < .001. (C) PRIMA-1^Met increased the expression of NOXA mRNA. OPM2 cells were treated overnight with 30 μM PRIMA-1^Met in the presence or absence of NAC (5 mM). The expression of NOXA mRNA was performed using quantitative reverse-transcription polymerase chain reaction with the TaqMan probe. The data represent 3 independent experiments. (D-E) Transient silencing of Noxa inhibited PRIMA-1^Met-induced cell death. OPM2 cells (B) or LP1 cells (C) were transfected with 100 pmol siCont or siNOXA (Life Technologies). At 72 hours, cells were treated for 24 hours with 100 μM PRIMA-1^Met, and cell death was assessed using flow cytometry (Apo2.7 staining). The data represent 4 independent experiments. Noxa expression was assessed using western blotting 72 hours after siRNA transfection.