Figure 6. Lapatinib and OSU-03012 interact to activate the extrinsic apoptosis pathway. (A) GBM6 and GBM12 cells were treated with vehicle (DMSO) or with lapatinib (1 μM) and OSU-03012 (1 μM). Cells were fixed at the indicated time points (5–24h) and the plasma membrane levels of CD95 determined by immunohistochemistry. Data are presented as the –Fold increase in CD95 density (n = 3, ± SEM). (B) GBM6 cells were either transfected with siRNA to knock down CD95 expression or were infected with a virus to express c-FLIP-s. Twenty four h later cells were treated with vehicle (DMSO) or with lapatinib (1 μM) and OSU-03012 (1 μM). Cells were isolated 24h later and viability determined by trypan blue exclusion assay (n = 3 ± SEM). (C) Cells were infected with empty vector virus (CMV) or with viruses to express dominant negative caspase 9, BCL-XL or c-FLIP-s. Twenty four h later cells were treated with vehicle (DMSO) or with lapatinib (1 μM) and/or OSU-03012 (1 μM). Cells were isolated 24h later and viability determined by trypan blue exclusion assay (n = 3 ± SEM). (D) GBM5 and GBM12 cells were transfected with empty vector plasmid (CMV) or with a plasmid to express MCL-1. Twenty four h later cells were treated with vehicle (DMSO) or with lapatinib (1 μM) and/or OSU-03012 (1 μM). Cells were isolated 24h later and viability determined by trypan blue exclusion assay (n = 3 ± SEM). (E) GBM12 cells were transfected with scrambled siRNA (siSCR) or siRNA molecules to knock down the expression of NOXA, PUMA, BIK, BCL-XL, MCL-1 and BCL-XL + MCL-1. Twenty four h later cells were treated with vehicle (DMSO) or with lapatinib (1 μM) and OSU-03012 (1 μM). Cells were isolated 24h later and viability determined by trypan blue exclusion assay (n = 3 ±SEM). (F) GBM12 cells were transfected with scrambled siRNA (siSCR) or an siRNA molecule to knock down the expression of AIF. Twenty four h later cells were treated with vehicle (DMSO) or with lapatinib (1 μM) and/or OSU-03012 (1 μM). Cells were isolated 24h later and viability determined by trypan blue exclusion assay (n = 3 ± SEM). *p < 0.05 less than corresponding value in siSCR cells.