Pc3 prostate cancer cells

786-O renal cancer cells, and PC3 prostate cancer cells were purchased from ATCC. RCC4, T47D, A549 and HCT-116 were purchased directly from ECACC. Unless used directly from a certified source, the identity of all cell lines was confirmed by STR genotyping and regularly tested for mycoplasma infection. RNA-seq analysis of RCC4 and 786-O cells also confirmed the presence of unique VHL gene coding mutations (chr3:10,183,725 C > G and chr3:10,183,841 G > del, respectively). Cell lines were grown in Dulbecco’s modified Eagle’s Medium, supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin and 10% fetal bovine serum (Sigma Aldrich). Primary renal cell cultures were generated from freshly excised ccRCC tissue and tumour-adjacent normal kidney. Briefly, tissue blocks were minced, incubated with 193U/ml Collagenase II and 3.33ug/ml DNase for one hour at 37 °C, with regular pipetting, and then filtered. Cell pellets were resuspended in growth medium (DMEM/F12 1:1, supplemented with glutamax, penicillin-streptomycin, insulin-transferrin-sodium selenite, 4 ng/ml triiodo-L-thyronine, 100 ng/ml epidermal growth factor, 36 ng/ml hydrocortisone and 10% foetal bovine serum). Experiments were performed after the 2^nd or 3^rd passage.
Hypoxic incubations were performed, as indicated, in an In Vivo2 400 Hypoxia Work Station (Ruskinn Technology).

PC3 prostate cancer cells were purchased from ATCC (Manassas, VA, USA). They were cultured in RPMI 1640 medium, which was supplemented with fetal bovine serum (10%), sodium pyruvate (1%), and penicillin-streptomycin (1% V/V, 100 U/mL final). Cells were grown in this supplemented culture medium in T-75 or T-150 flasks and incubated at 37 °C and 5% CO₂. Cells were passaged at ~90% confluence; medium was removed from flasks, the flasks washed with Hanks Balanced Salt Solution, and cells incubated in cold 0.05% trypsin-EDTA for 9–10 min before being split (1:16) into new flasks. Cells were counted with a hemocytometer and trypan blue stain when specific cell numbers were needed.

PC-3 prostate cancer cells were purchased from ATCC, and maintained in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (PAA). PC-3M-luc-2 were purchased from Caliper Life Sciences (Buckinghamshire, UK) and maintained in RPMI-1640 (Life Technologies) supplemented with 10% fetal bovine serum (PAA). Cells were cultivated in 175 cm² flasks in a humidified incubator; once 80–90% confluency was reached, cells were passed to maintain exponential growth. Mycoplasma absence was confirmed monthly, using Plasmotest (Invivogen, France).