HER-2 levels and effects on cell viability of T-DM1, trastuzumab and vinorelbine in NSCLC cell lines. (A) Densitometric quantification of total HER-2 protein level, detected by Western blotting, was calculated using Quantity One software. Three different Western blot experiments were performed on total cell lysate of the indicated NSCLC cell lines. A representative Western blot analysis is reported as inset. (B) HER-2 protein levels on cell surface was quantified by flow-cytometry and expressed as molecular equivalent of fluorochrome (MEF) as described in Methods section. (C) Calu-3, Calu-6, H3255, H1781 and H322 cells were exposed to increasing concentrations (0.001, 0.01, 0.1, 1 and 10 μg/ml) for 72 h and then cell viability was assessed by MTT assay. (D) Percent of inhibition of cell viability induced by T-DM1 at 1 μg/ml as a function of HER-2 level (E) Calu-3 and Calu-6 cell viability inhibition curves after treatment with increasing vinorelbine concentrations. Data are expressed as mean + SD of three different experiments.