Effects of antioxidants on ROS generation, apoptosis, and cell-signaling proteins induced by coadministration of 2-ME and the HDACIs, NaB or SAHA. (A) U937 cells were exposed to 2-ME (0.5 μM) combined with either NaB (1 mM) or SAHA (1.5 μM) for 6 hours, after which total cellular extracts were prepared and subjected to Western blot analysis using antibodies against GPx1, Cu/Zn-SOD, MnSOD, and Trx. (B) U937 cells were pretreated with antioxidants including TBAP (200 μM), catalase (5000 U/mL), or sodium formate (SF, 2 mM) for 2 hours, followed by the addition of 2-ME (0.5 μM) in the presence or absence of NaB (1 mM) or SAHA (1.5 μM) for 6 hours. Cells were stained with DCFH-DA, after which ROS production was analyzed by flow cytometry as described in “Materials and methods.” (C) U937 cells were pretreated with antioxidants including TBAP (200 μM), catalase (5000 U/mL), or sodium formate (SF, 2 mM) for 2 hours, followed by the addition of 2-ME (0.5 μM) in the presence of NaB or SAHA for 24 hours, after which cells were stained with annexin V and PI and apoptosis was analyzed by flow cytometry as described in “Materials and methods.” ^*Values for cells treated with 2-ME and an antioxidant (TBAP or catalase) in the presence of NaB or SAHA are significantly less than those obtained for cells treated with 2-ME plus either NaB or SAHA in the absence of the antioxidant (Student t test; P < .01). U937 cells were pretreated with antioxidants including TBAP (200 μM), catalase (5000 U/mL), or sodium formate (SF, 2 mM) for 2 hours, followed by the addition of 2-ME (0.5 μM) in the presence of NaB or SAHA for 24 hours, after which total cellular or cytosolic extracts were prepared and subjected to Western blot analysis using antibodies against PARP, cleaved caspase-3, full-length caspase-8 and caspase-9, AIF, cytochrome c (D) or cell-signaling proteins including phospho-Akt, Akt, phospho-JNK, and JNK (E). (F) U937 cells were stably transfected with constitutively active forms of Akt (CA-3) or an empty vector (pUSE). Cells were treated with 0.5 μM 2-ME in the presence of NaB (1 mM) or SAHA (1.5 μM) for 6 hours, after which cells were stained with DCFH-DA, and ROS production was analyzed using flow cytometry as described in “Materials and methods.” For panels A, D, and E, each lane was loaded with 30 μg protein. Blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading and transfer. Two additional studies yielded equivalent results. For panels B, C, and F, results represent the means ± SD for 3 separate experiments performed in triplicate.