Growth factor supplements

Primary human prostatic stromal cells were generated from ethically obtained tissue collected by the Wales Cancer Bank, from informed and fully consented patients. Cells were isolated from radical retropubic prostatectomy cores, taken from sites of palpable disease and also from apparently normal tissue from the opposite side of the same prostate. Representative cores were stained (H&E), and confirmed by an independent pathologist as cancerous stroma or normal respectively. Homogenized tissue was collagenase I treated (200U/ml; Lonza, Wokingham, UK) and liberated cells plated in stromal cell basal medium (SCBM) (Lonza), which selects for stromal cell types. Cultures were left undisturbed for 7-10 days. At first harvest, cells were subsequently maintained in DMEM:F12 media (Lonza). These cultures were confirmed free of epithelial cells by immuno-fluorescence staining demonstrating lack of cytokeratins, and used in experiments at passage 4-6. Endothelial cells, of human umbilical chord origin, were purchased from Lonza, and maintained in EBM2-media with growth factor supplements (Lonza). For the angiogenesis assay supplements were withdrawn 24h before the experiment, and remained withdrawn for the duration as described [26 (link)].

Human umbilical vein endothelial cells (HUVEC) (ThermoFisher Scientific, Cat# C0035C), certified by the supplier to be free of mycoplasma and pathogens, were cultured in EGM2 complete medium (Lonza, Cat# CC-3156) along with growth factor supplements (Lonza, Cat# CC-4176) at 37°C in 5% CO₂ in a humidified atmosphere. Cells at less than 4–5 passages were used for all experiments. For siRNA-mediated knockdown, HUVECs were transfected using the Lipofectamine RNAiMAX (Invitrogen, Cat# 13778075) with target gene-specific siRNA target sequences or control siRNA (Negative Control #1 siRNA (Cat#AM4611); YAP1 #1: (5’−3’) GGUGAUACUAUCAACCAAAtt (ID:s20366); YAP1 #2: (5’−3’) ACAGUCUUCUUUUGAGAUAtt (ID:s20367); TAZ (WWTR1) #1: (5’−3’) GUACUUCCUCAAUCACAUAtt (ID:s24789); TAZ (WWTR1) #2: (5’−3’) GGAUACAGGAGAAAACGCAtt (ID:s24787); HIF1A #1: (5’−3’) CCAUAUAGAGAUACUCAAAtt (ID:s6539) from ThermoFisher Scientific. For hypoxia experiments, siRNA-transfected cells were grown in 1% O₂ and 5% CO₂ at 37°C for 24 hr in a humidified atmosphere.