Dmem high glucose medium

Human glioblastoma cell lines U373 (cell line listed under catalogue number 89,081,403 has been re-named as U-251) and U87 were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and were cultured in DMEM high glucose medium (GE Healthcare, Chicago, IL, USA), supplemented with 10% (v/v) FBS, 2 mM l-glutamine, 100 IU/mL penicillin and 100 µg streptomycin. Glioblastoma stem cell line NCH421k was obtained from CLS (Cell Lines Service GmbH, Eppelheim, Germany) and expanded as spheroid suspensions in complete Neurobasal Medium (Invitrogen, Life Technologies, Carlsbad, CA, USA) containing 2 mM l-glutamine, 1 × penicillin/streptomycin, 1 × B-27 supplement (Invitrogen, Life Technologies, Carlsbad, CA, USA), 1 U/mL heparin (Sigma-Aldrich, St. Louis, MO, USA), 20 ng/mL bFGF and EGF (both from Invitrogen, Life Technologies, Carlsbad, CA, USA). Cells were transfected with the plasmid vector pCMVDsRed-Express2 and pEGFP-N1 to stably express the red fluorescent protein DsRed and enhanced green fluorescent protein eGFP, as described previously [59 (link),63 (link)]. All cell lines were maintained at 37 °C with 5% CO₂ and 95% humidity. All cell cultures were tested for mycoplasma contamination using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel, Switzerland).

Human embryonic kidney (HEK) 239T cells were cultured in DMEM high glucose medium (GE Healthcare Life Science) supplemented with 10% FBS and 100U/ml streptomycin/penicillin. Human primary cells were isolated as previously described [11 , 12 (link)]. Cell purity was > 95% as determined by post-FACS analysis (Figure S1).

The human ccRCC cell lines (RCC4, RCC10 and 786.0) and their isogenic counterparts stably expressing VHL (RCC4/VHL, RCC10/VHL and 786.0/VHL) were kindly provided by Amato J. Giaccia (Stanford University, CA, USA). All cell lines were tested for mycoplasma and authentication was performed by short tandem repeat (STR) DNA profile at Genetica DNA Laboratories (Burlington, NC, USA). Cells were maintained in DMEM/high glucose medium (GE Healthcare Life Science, UT, USA), supplemented with 10% Fetal bovine serum (FBS) (Wisent Bio Science, QC, Canada), 2 mM L-glutamine and 1mM sodium pyruvate (GE Health Life Sciences, ON, Canada) and cultured at 37˚C in a humidified incubator with 5% CO₂.

A CCK8 kit, doxorubicin hydrochloride, and the (6-maleimidocaproyl) hydrazone of DOX (DOXO-EMCH) were purchased from Med Chem Express LLC Co., Ltd. (Shanghai, China). DMEM/high glucose medium and trypsin were procured from GE Healthcare Life Sciences, while female BALB/c nude mice (8 weeks old) were obtained from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All other chemicals utilized in this study were purchased from J&K Scientific Ltd.