Cell culture

Human glioblastoma U87 MG (HTB-14^™), murine melanoma B16-F10 (CRL-6475^™) and canine osteosarcoma D17 (CCL-183^™) cell lines were purchased from ATCC^® (American Type Culture Collection). Human breast cancer MDA-MB231 was a generous gift from Prof. E. Dahl (Hospital of the RWTH Aachen University). Canine oral melanoma 17CM98 were kindly provided by Prof. Dr. David Vail, University of Wisconsin-Madison. Canine soft-tissue sarcoma K9STS and hemangiosarcoma HAS were kindly provided by Prof Dr. Carla Rohrer-Bley, Vetsuisse Faculty Zurich. Human hepatoma (Hep3B), melanoma (501), renal clear cell carcinoma with (i.e., RCC4) and without (i.e. VHL reverted cell line: RCC4/VHL) loss-of-function mutation in the VHL tumor suppressor gene as well as breast carcinoma (MCF7, MDA-MB468) cells were available as laboratory stocks from previous works [57, 58]. HEP3B, U87, RCC4 and RCC4/VHL, MCF7, MDA-MB468 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM), B16F10 cells in Eagle’s minimum essential medium (EMEM) and 501 melanoma cells in RPMI medium (RPMI Media 1640). Each medium contained high glucose levels (4.5g/l), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. Selection pressure in RCC4 and RCC4/VHL cultures was maintained by addition of G418 antibiotic (0.5mg/ml). Canine cell lines (D17, 17CM98, K9STS and HAS) were cultured in RPMI medium (RPMI Media 1640) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 1% HEPES, 1% sodium pyruvate, 1% non-essential amino acid (NEAA) and 1% GlutaMax. All cell lines were kept at 37°C in humidified air with 5% CO₂ and a pO₂ of 141.6 mmHg ([O₂] = 18.6% O₂).