Figure 4. Regulation of lapatinib + obatoclax lethality by toxic BH3 domain proteins. (A) BT474 (wild type) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down BAK, BAK, NOXA, and PUMA, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) ^#P < 0.05 less than corresponding value in siSCR cells; ^##P < 0.05 less than corresponding single knock down cells. (B) BT474 (WT, wild type; AR, anoikis-resistant) cells were transfected with empty vector plasmid (CMV) or plasmids to express BAX, BAK, NOXA, and PUMA as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in CMV cells; **P < 0.05 greater than corresponding single expression cells. (C) BT474 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1, c-FLIP-s, and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; ^%P < 0.05 less than corresponding value in siMCL-1 cells. (D) SKBR3 (AR, anoikis-resistant) cells were transfected with scrambled siRNA (siSCR, 20 nM) or siRNA molecules to knock down MCL-1 and BCL-XL, as indicated. Thirty-six hours after transfection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) *P < 0.05 greater than corresponding value in siSCR cells; ^%P < 0.05 less than corresponding value in siMCL-1 cells. (E) BT474 (AR, anoikis-resistant) cells were infected with empty vector adenovirus (CMV) or with adenoviruses to express dominant negative MEK1 or dominant negative AKT, as indicated. Thirty-six hours after infection cells were treated with vehicle (VEH, DMSO) or with lapatinib (lap, 1 μM) and obatoclax (GX, 50 nM). Cells were isolated 12 h later and viability determined by trypan blue (± SEM, n = 3) ^#P < 0.05 less than corresponding value in CMV cells.