Effect of T-DM1 on EGFR-mutant PC9 cell line become resistant to gefitinib for HER-2 overexpression. (A) PC9 and PC9/HER2 c1 cells were exposed to increasing concentrations of T-DM1 for 72 h and then cell viability was assessed by MTT assay. Data are expressed as mean + SD of three different experiments. (B) Immunoblot analysis of proteins of signalling transduction pathways were conducted on cell lysates obtained after treatment with T-DM1 (1 μg/ml) for 24 or 48 h. (C) Curves of growth inhibitory effects of gefitinib and combined treatment gefitinib plus T-DM1 versus theoretical Bliss additivity curve are reported. Cells were treated with the drugs for 72 h and then cell number was assessed by MTT assy. Data are expressed as percent inhibition of cell proliferation versus control cells. The experiments, repeated three times, yielded similar results.