Mig6 overexpression synergistically enhances the cell growth inhibitory effect of gefitinib. (A) EGFR phosphorylation influenced by Mig6 overexpression in the presence of gefitinib. Phosphorylation of EGFR in H1299EGFR-WT cells (green circle) and H1299EGFR-WT cells with overexpressed Mig6 (red square) was measured 5 minutes after EGF (10 nM) administration, in the presence of gefitinib at the indicated concentrations. Western blots were performed twice, and the mean values of the signal intensities were calculated. The mean values were normalized so that the values without gefitinib were equal to 1. (B) Cell growth influenced by Mig6 overexpression in the presence of gefitinib. Cell viabilities of H1299EGFR-WT cells (left panel) and H1299EGFR-WT cells with overexpressed Mig6 (right panel) were measured by an MTT assay 3 days after stimulation without (blue circle) or with (red square) 10 nM EGF in the presence of various doses of gefitinib (0, 0.1, 0.5, 1, and 5 μM). The data shown are the means and the standard deviations of triplicated or quadruplicated experiments. (C) Inhibitory effect of a combination of Mig6 and gefitinib (Mig6 & gefitinib pair). Red squares show concentrations of ERKPP at t = 5 minutes under paired perturbations in silico, plotted against inhibitory intensities X (0, 10, 20, 30, 40, 50, 60, 70, 80, and 90). See Materials and Methods section for the explanation of X. The blue line shows the effect expected from a linear interpolation of two individual perturbations (additive effect line). The area above the additive line shows a smaller effect than the additive effect (antagonistic effect zone), and the area below it shows a stronger than additive effect (synergistic effect zone).