Pher3

5 μm sections from FFPE tissue were mounted on glass slides, air dried at 56°C overnight and subjected to immunostaining using an automated platform (Ventana Discovery XT, Tucson, AZ, USA). The following primary antibodies and dilutions were used: pERK1/2 1:300, pAkt(Ser473) 1:30, pEGFR 1:110, pmTOR 1:130, pHer3 1:70 (all from Cell Signaling Technology Inc., Danvers, MA, USA). After staining, sections were treated with ascending ethanol concentrations and xylene and were finally covered with Pertex (Medite GmbH, Burgdorf, Germany). Sections were examined under a light microscope by a pathologist. Tumor cell staining was classified as absent, weak, moderate, or strong and a staining score was calculated based on the extent of staining according to the formula: score = 3 × percentage of strongly stained tumor cells + 2 × percentage of moderately stained tumor cells + 1 × percentage of weakly stained tumor cells. All IHC staining protocols used within this study are “fit for purpose” and validated according to FDA guidelines, and staining procedures were also conducted according to standard operating procedures. Therefore, assays are controlled in terms of specificity and accuracy.

Gefitinib (Iressa, TOCRIS, UK) was dissolved in dimethyl sulfoxide (DMSO, Sigma) prior to the experiments. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Amresco, USA) and propidium iodide (PI) were dissolved in PBS to final concentrations of 5 mg/ml and 50 μg/ml, respectively. The Annexin V-APC apoptosis detection kit was obtained from BD Biosciences (BD, NY, USA). Antibodies against HER3, p-HER3, HER2, p-HER2, AKT, p-AKT, ERK, p-ERK and Cyclin B1 were purchased from Cell Signalling Technology (Beverly, MA, USA), and the antibody against β-actin was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).