Effects of 2-ME combined with the HDACIs, NaB and SAHA, on apoptosis-related gene expression and various signal-transduction pathways. (A) U937 cells were treated with 2-ME (0.5 μM) in presence or absence of NaB (1 mM) or SAHA (1.5 μM) for 24 hours, after which total cellular extracts were prepared and subjected to Western blot assay using antibodies against PARP, caspase-8, caspase-9, and cleaved caspase-3. Cytosolic extracts were also prepared and subjected to Western blot assay using antibody against AIF and cytochrome c. (B) U937 cells were exposed to 2-ME (0.5 μM) combined with either NaB (1 mM) or SAHA (1.5 μM), after which total cellular extracts were prepared and subjected to Western blot assay using antibodies against p21, p27, Bcl-2, Bcl-x_L, Bad, Bid, and bax. Cytosolic and membrane extracts were also prepared and subjected to Western blot assay using antibody against Bax. (C) After coadministration of 2-ME and NaB or SAHA for 24 hours, total cellular extracts were prepared and subjected to Western blot assay using antibodies against phospho-Akt, Akt, phospho-ERK, ERK, phospho-p38, p38, phospho-JNK, and JNK. (D) U937 cells were exposed to 2-ME (0.5 μM) combined with either NaB (1 mM) or SAHA (1.5 μM) for the indicated exposure interval, after which total cellular extracts were prepared and subjected to Western blot assay using antibodies against PARP, phosphor-Akt, Akt, phosphor-JNK, and JNK. (E) U937 cells were pretreated with the caspase inhibitor Z-VAD-FMK (10 μM) for 1 hour, followed by cotreatment with 0.5 μM 2-ME and 1 mM NaB or 1.5 μM SAHA for 24 hours, after which total cellular extracts were prepared and subjected to Western blot assay using antibodies against PARP, cleaved caspase-3, caspase-8, phosphor-Akt, Akt, phosphor-JNK, and JNK. For Western blot assay, 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.