The effect of knockdown of EZH2 or ectopic expression of TXNIP or knockdown of TXNIP in MOLM-14 cells. (A) MOLM-14 cells were transduced with pSIREN-EZH2 shRNA or nontargeting virus for 72 hours. The protein levels of EZH2, TXNIP, and H3K27me3 were determined by Western blot analysis. (i) β-actin was used as the loading control; the mRNA levels of EZH2 and TXNIP were analyzed by (ii) RT-PCR and (iii) RQ-PCR. Densitometric analysis was performed using Amersham Image Scanner with LabScan ImageQuant TL software. The expression ratio of EZH2, and TXNIP was calculated as the EZH2-shRNA–treated samples relative to control samples after normalization with respective β-actin level. (B) MOLM-14 cells were transfected with pLVX-TXNIP or control lentivirus (Mock) for 72 hours, followed by Western blot analysis of TXNIP and FACS analysis of ROS production. (C) Trypan blue exclusion method was used to determine the cell viability in a hemocytometer under an invert microscope. (D-F) MOLM-14 cells were transduced with scramble- (left panels) or TXNIP-shRNA (right panels). Cells were then incubated with either DZNep 0, 1, 2μM, followed by Western blot analysis of TXNIP protein (D), and apoptosis after 48 hours (F), or DZNep 2μM, followed by quantification of ROS production at 0, 8, 16, and 24 hours (E). Three separate determinations were indicated as mean ± SD.