45 MET immunoreactivity and poor prognosis coring. In total, six tissue cores (0.6 mm diameter) were sampled from each cancer; three from the center and three from the periphery. Using a microtome, 4 μm thick slices were cut from the FFPE cancer tissue and TMA blocks that were processed for immunohistochemistry. MET antibody validation cell line panel and culture conditions Based on western blot results retrieved from the literature (21, 36-38) and datasheets provided with the purchased MET antibodies, a MET antibody validation cell line panel was developed. The panel consists of the following cell lines: LNCaP, SK-BR-3 (p145MET negative according to the retrieved information), HeLa, HT-29, PC3, DU145 (p145MET positive according to the retrieved information) and DU145#Sh167 (a MET silenced cell line). DU145#Sh167 was derived from DU145 by means of lentiviral infection as previously described (39). Additional information is provided in Supplementary table S1. All cell lines were obtained from different departments within the Erasmus Medical Center (EMC, Rotterdam, The Netherlands). Cells were cultured at 37°C under 5% CO2 in DMEM/F12 (Life Technologies™) or RPMI 1640 medium (Life Technologies™), supplemented with varying percentages of Fetal Calf Serum (Life Technologies™) and 1% Penicillin/Streptomycin (Life Technologies™). Only DU145#Sh167 was grown in the presence of Puromycin (Sigma-Aldrich®; Zwijndrecht, The Netherlands). More information is indicated in Supplementary table S2. For all experiments (i.e., quantitative real-time PCR, western blot analysis, immunocytochemistry and immunohistochemistry) the cell lines were cultured as biological duplicates in view of independent validation of the results. RNA isolation, cDNA generation and quantitative real-time PCR Cells were cultured in T25 flasks until 75-90% confluence was reached and harvested in TRIZol® Reagent (Life Technologies™/Ambion®) for totRNA isolation according to the manufacturer’s protocol. In total, 25 μg of totRNA was treated with RNase-free DNaseI (Life Technologies™/Ambion®) for 60 minutes at 37°C. Next, the totRNA was purified using the RNeasy mini kit (Qiagen; Venlo, The Netherlands) and eluted in 30 μL of DEPC treated H2O. Subsequently, cDNA was synthesized using SuperScript ® II Reverse Transcriptase (Life Technologies™/Invitrogen™) in accordance with the manufacturer’s instructions. 3
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