R3 igf

CGMTM Chondrocyte Growth BulletKitTM containing fetal bovine serum (FBS), GA-1000, R3-IGF, bFGF, transferrin, and insulin were provided by Lonza (Walkersville, MD, USA). A high-capacity RNA-to-cDNA kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The ReliaPrep RNA Cell Miniprep system, ROS-Glo H₂O₂ assay, Caspase-Glo 3/7 assay, and Caspase-Glo 1 inflammasome assay were provided by Promega (Fitchburg, WI, USA). A radioimmunoprecipitation-assay (RIPA) lysis buffer and BCA protein-assay kit were from Thermo Fisher Scientific. SigmaFast BCIP/NBT reagent and molecular-grade purity water were provided by Sigma-Aldrich (St Louis, MO, USA). The polyclonal (mouse) anti-KDEL antibody was purchased from Enzo Biochem (Farmingdale, NY, USA) and monoclonal (mouse) anti-HIF1α antibody from BD Biosciences (San Jose, CA, USA). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG was from Rockland Immunochemicals (Limerick, PA, USA). HRP-conjugated anti-rabbit IgG antibody, polyclonal (rabbit) anti-β-tubulin antibody, monoclonal (mouse) anti-CHOP antibody, monoclonal (mouse) anti-XBP-1s antibody, polyclonal (rabbit) anti-ORP150 antibody, and SignalFire Elite ECL Reagent were provided by Cell Signaling Technology (Boston, MA, USA). Tauroursodeoxycholic acid (TUDCA), hyaluronic acid (HA), and tunicamycin (TNC) were purchased from EMD Millipore corporation (Temecula, CA, USA).

For EV collection, ASC/hTERT1 (Evercyte, Vienna, Austria) cells were cultivated using Endothelial Cell Growth Medium-2 medium (Lonza, Basel, Switzerland) supplemented with 2% FBS (Gibco, Thermo Fisher Scientific, Bleiswijk, The Netherlands), and hydrocortisone, hFGF-B, VEGF, R3-IGF, Ascorbic Acid, hEGF, Heparin, GA-1000 (Lonza, Basel, Switzerland). For in vivo and in ovo experiments, GBM cell lines were cultivated in DMEM/F12 + Glutamax + 10% FBS (Gibco, Thermo Fisher Scientific, Bleiswijk, The Netherlands) for HROG36 (Cell Lines Service GmbH, Eppelheim, Germany); DMEM + Glutamax + 10% FBS (Gibco, Thermo Fisher Scientific, Bleiswijk, The Netherlands) for U87 MG (European Collection of Cell Cultures (ECACC, Salisbury, UK); aMEM + Glutamax + 10% FBS (Gibco, Thermo Fisher Scientific, Bleiswijk, The Netherlands) for T98G (ATCC, Manassas, VA, USA) cells. All mediums were supplemented with penicillin–streptomycin solution (Gibco, Thermo Fisher Scientific, Bleiswijk, The Netherlands). All cells were cultured in a 37 °C, 5% CO₂ humidified incubator.

Conditionally immortalised GECs (obtained from the University of Bristol) were cultivated using endothelial cell growth medium (EBM-2, Lonza) supplemented with 5% FCS and growth factors (hFGF, R3-IGF, hEGF), GA-1000, ascorbic acid and hydrocortisone (Lonza). Conditionally immortalised podocytes (obtained from the University of Bristol) were cultivated in 10 cm dishes in RPMI-1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS (Gibco), 1 g/L glucose (Roth), 2 g/L sodium hydrogen carbonate (Roth), insulin-transferrin-selenium (Gibco) and 200 U/mL penicillin/streptomycin (Gibco). To maintain proliferation, cultures were kept at 33 °C, 5% CO₂ and 95% relative humidity. After reaching around 80% confluence, the cells were passaged using 0.25% Trypsin/EDTA (Gibco), and experiments were performed on cells in passages 2–4 after thawing. Medium was changed every 2–3 days.