The role of eIF2-α phosphorylation in cell death induced by OSU-03012 and lapatinib. A-B: MDA-MB-231 (A) or BT474 (B) cells (5 x 105) were transfected with the indicated siRNA molecules and incubated for 48 h. Cells were then treated with either vehicle (Veh) or the combination of OSU-03012 (2 μM) and lapatinib (2 μM) (combo) as indicated and either subjected immunoassays (bottom panels) or plated for clonogenic capacity (top graphs). Numbers indicated are percent control (e.g. Veh). C-D: MDA-MB-231 cells (5 x 105) were transfected with a control vector (Vector), wild-type (WT) or the Ser51Ala mutant (S51A) eIF2-α plasmids. After a further 24 h cells were plated onto 6-well plates to assay for clonogenic capacity (D) or subjected to immunoblotting as described in Materials and Methods (C). Cells were treated with either vehicle (Veh, DMSO) or OSU-03012 (2 μM) and lapatinib (2 μM) in combination (combo) for 24 h (D) or 3 h (C). Colonies were allowed to develop for the next 10-14 days. * indicates a p < 0.05 with respect to vehicle-treated cells. E: MDA-MB-231 cells (1 x 106) were plated and treated with the indicated drugs (Vehicle (Ctr, DMSO), OSU-03012 (OSU, 2 μM), lapatinib (Lap, 2 μM) or the combination (Combo)). Three hours later, cells were harvested and subjected to immunoblotting. Samples were probed with the indicated antibodies (see Materials and Methods).