RT�CPCR analysis for EGFR, VEGF, and VEGFR-2 DZNeP Total RNA and genomic DNA were extracted from the four cell lines. Total RNA of 1��g was converted into cDNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche, Basel, Switzerland) in accordance with the manufacturer’s instructions. mRNA expression of EGFR, VEGF, and VEGFR-2 was assessed by the RT�CPCR method. Quantitative real-time PCR was conducted using LightCycler480 (Roche) in accordance with the manufacturer’s instructions. TaqMan Probes (Applied Biosystems, Foster City, CA, USA) for EGFR and VEGF were used. For standardisation of the amount of RNA, expression of glyceraldehyde-3-phosphate dehydrogenase in each sample was quantified. Primers are shown in the Supplementary Table 1.

Mutation analysis of the EGFR and KRAS genes For the sequence analysis of EGFR and KRAS, cDNA or genomic DNA was sequenced after PCR amplification. Direct sequencing was conducted using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and analysed on an ABI Prism 3100 Sequencer (Applied Biosystems). Primers are shown in the Supplementary Table 1. Immunohistochemistry Tissue preparation and immunohistochemistry (IHC) were conducted as reported earlier (Yoshikawa et al, 2008). A polymer-based method (Envision+Dual Link System-HRP; Dako, Glostrup, Denmark) was used for EGFR, VEGF, and Ki67 staining, and a standard ABC method (Vectastain Elite ABC kit; Vector Laboratories, Burlingame, CA, USA) was used for CD34 staining.

Sources and dilutions of primary antibodies were as follows: mouse anti-human EGFR (1:100 dilution, clone 31G7; Zymed, South San Francisco, CA, USA), rabbit anti-human VEGF (1:50 dilution; Zymed), mouse anti-human Ki67 (1:100 dilution, clone Ki-S5; Chemicon, Temecula, CA, USA), and rat anti-mouse CD34 (1:50 dilution, clone MEC14.7; Abcam, Cambridge, UK). Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) was conducted to assess the degree of apoptosis by using an In Situ Cell Death Detection Kit, POD (Roche) in accordance with the manufacturer’s instructions. Fluorescence in situ hybridisation for the EGFR gene locus EGFR gene copy number per cell was investigated by fluorescence in situ hybridisation (FISH) using the LSI EGFR SpectrumOrange/CEP7 SpectrumGreen probe (Vysis, Downers Grove, IL, USA), in accordance with a published protocol (Ooi et al, 2004).

Positivity for gene amplification was defined as the presence of clustered signals or 4 copies of orange signals. Drug and formulation Vandetanib was provided Brefeldin_A by AstraZeneca (Macclesfield, UK). For the in vitro study, vandetanib was formulated as a 10-mM stock in 100% dimethylsulphoxide and stored at ?20��C. Just before in vitro use, the stock solution was diluted in culture medium to the required concentration.