Recombinant human tweak

Human Embryonic Kidney 293 cells (HEK293) and Human umbilical vein endothelial cells (HUVECs) were obtained from ATCC (Molsheim, France) and were respectively cultivated in DMEM with 10 % FCS and 1 % penicillin–streptomycin (Invitrogen, Illkirch, France) and EBM-2 basal medium supplemented with EGM-Plus bullet kit (Lonza, Basel, Switzerland). Recombinant human TWEAK was purchased from Peprotech, Neuilly-sur-Seine, France.

U-87 MG (ATCC) and U-251 MG (Sigma-Aldrich) (formerly known as U-373 MG) (ECACC 09063001) GBM cell lines were kindly gifted from Prof. Phillip Koeffler at the Cancer Science Institute, Singapore^{47 (link)}. Cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM; HyClone) supplemented with 10% fetal bovine serum (FBS; Sigma) and regularly tested negative for mycoplasma contamination using Mycoplasma PCR Detection Kit (Applied Biological Materials Inc; G238). Cell lines were not authenticated via STR profiling by us. Recombinant human TWEAK (PeproTech; 310-06) was reconstituted according to the manufacturer’s recommendations. All GBM cell lines were treated with 10 ng/ml of TWEAK.

The prostate cancer cell lines PC-3 (androgen-insensitive) and LNCaP (androgen-sensitive) and the histologically normal prostate epithelial RWPE-1 cell line (immortalized with papilloma virus 18) were purchased from Sigma-Aldrich (Barcelona, Spain). PC-3 cells were cultured in Ham’s F-12K (Kaighn’s) Medium (1:1 mixture) with L-glutamate (Invitrogen/Gibco, Fisher Scientific SL, Madrid, Spain). LNCaP cells were cultured in RPMI 1640 medium (Merck KGaA, Darmstadt, Germany) supplemented with 1-mM sodium pyruvate (Gibco). PC-3 and LNCaP cultures were supplemented with 10% fetal bovine serum. RWPE-1 cells were cultured in keratinocyte serum-free medium plus 5 μg/mL bovine pituitary extract. All the cells were supplemented with 1× antibiotic-antimycotic (Gibco) and 5 μg/mL Plasmocin^TM (InvivoGen, San Diego, CA, USA). Where indicated, the cells were grown in serum-deprived medium overnight before stimulation for 24 or 48 h with 100 ng/mL human recombinant TWEAK (PeproTech, bioNova cientifica, Barcelona, Spain) and were cultured in a humidified 5% CO₂ atmosphere at 37 °C.