Combined treatment with LY294002, PD98059 and arsenite induces TRAIL-mediated apoptosis in LU1205 melanoma cells. (a) Herbimycin (20 μM), but not AG1478 (20 μM), enhances arsenite-induced apoptosis in LU1205 melanoma cells; combination of LY294002 (50 μM) and PD98059 (50 μM) also upregulates arsenite-induced apoptosis. Cell-cycle apoptosis analysis was performed using flow cytometry of PI-stained cells. (b) Western blot analysis of total and phospho-AKT, phospho-ERK and TRAIL levelsfollowing LY294002 (50 μM) and PD98059 (50 μM) treatment in the presence or absence of arsenite (10 μM). NF-κB DNA-binding activity of nuclear proteinswas determined by EMSA. Two DNA-binding complexesare indicated NF-κB p65-p50 and (p50)2. (c) Apoptosis analysis of LU1205 cells treated by LY294002 (50 μM) þ PD98059 (50 μM) with or without arsenite (10 μM) in the presence of nonspecific IgG, inhibitory anti-TRAIL or anti-TNFα mAb (5 mg/ml). FACS analysis was performed with PI-stained melanoma cells. A percentage of apoptotic cells 24 h after treatment is indicated