Cell lines and lentiviral vectors Clausole campione

Cell lines and lentiviral vectors. For this study we used different MPM cell lines: REN cells for Epithelioid subtype, MM98 for Sarcomatous subtype and MSTO-211H for Biphasic subtype. Met-5A (ATCC® CRL-- 9444™) are SV40 immortalized non-tumorigenic mesothelial cells. REN and MM98 cells were grown in DMEM (Lonza), MSTO-211H were grown in RPMI1640 (Lonza) and Met-5A cells in Medium 199 (Life Tech). All media were supplemented with 10% FBS (Fetal Bovine Serum) and 1% penicillin, streptomycin, L-glutamine, and all cells were maintained at 37°C and 5% CO2. For Western Blotting analysis, normal human primary mesothelial cells were used as Non-tumoral control. Cells were purchased from Cambridge Bioscience (Cambridge, UK) and maintained according to manufacturer instructions, up to 3 passages. Densitometric analysis was performed by ImageJ software. MPM cells were stably infected with either one constitutive lentiviral vector carrying scramble ShRNA, used as control, or one carrying eIF6 ShRNA. Lentiviral vectors, pGIPZ Lentiviral ShRNA, were provided by Open Biosystem. Specifically, mature antisense sequences of constitutive shRNA of eIF6 were: 5'-AGCTTCCTACTAGCACCTG-3' (V3LMM_421640; GIPZ eIF6 shRNA: RMM4532-EG16418). After lentiviral infection, REN cells were selected with puromycin (1µg/ml) for 48 hours, expanded and treated with Enzastaurin as specifically described.