Figure 5.

Inhibition of phosphoinositide 3-kinase (PI3K)/Akt by LY294002 (LY) enhanced phenethyl isothiocyanate (PEITC)-induced apoptosis in human leukemia cells. U937 cells were pretreated with 20 μM of LY for 1 h, followed by the addition of 4 μM of PEITC for 3 and 6 h. (a) Cells were stained with Annexin V/propidium iodide (PI), and apoptosis was determined using flow cytometry as described in Materials and Methods. The values obtained from Annexin V/PI assays represent the means±standard deviation (S.D.) for three separate experiments. ^**Values for cells treated with PEITC and LY in combination were significantly greater than those for cells treated with PEITC alone by Student's t-test; P<0.01. Total cellular extracts were prepared as described in Materials and Methods, and subjected to western blot analysis using antibodies against apoptosis-related proteins, including poly(ADP-ribose) polymerase (PARP), cleaved-caspase-3 (C-Caspase-3), caspase-8, and C-Caspase-9 (b), antiapoptotic protein Mcl-1 (c), and cell signaling molecules, including phospho-Akt, Akt, phospho-Jun N-terminal kinase (JNK), and JNK (d). For western blot assay, each lane was loaded with 30 μg of protein; blots were subsequently stripped and reprobed with antibody against β-actin to ensure equivalent loading. Two additional studies yielded equivalent results