DZNep treatment increased TXNIP expression and ROS production. TXNIP expression was determined by RT-PCR and Western blot analysis in MOLM-14 (A) and KG-1 (B) cells treated with either DMSO control or DZNep for 24 hours. (C) Trx activity in MOLM-14 cells treated with DMSO control or 2μM DZNep for 48 hours was measured at 412nM. Three separate measurements were indicated as mean ± SD. (D) FACS analysis of ROS production in MOLM-14 cells treated with DMSO control, DZNep, or NAC + DZNep. (E) Western blot analysis of cleaved PARP. (F) MOLM-14 cells were treated with DMSO control, DZNep 1μM or 2μM for 48 hours with or without NAC. Cells were washed, lysed, and subjected to Western blot analysis of PERK. β-actin was used as a loading control. (G) One representative FACS profile of ROS production in one CD34⁺CD38⁻ leukemia early progenitor cells treated with DZNep. (H) FACS analysis of ROS production (left panel) and apoptosis (right panel) in MOLM-14 cells treated with DZNep 2μM in a time-dependent manner. The amount of ROS production and apoptosis in treated samples were normalized to that of baseline in the control samples (0 hours). Three separate measurements were indicated as mean ± SD. Arrows indicate the starting time point when ROS and apoptosis were significantly increased, respectively.