Phtpp

In preparation for cell proliferation assays, cells were cultured at a final cell number of 50,000 cells/ ml in phenol red-free RPMI media (Sigma-Aldrich, Poole, UK) to eliminate the weak oestrogenic effect of this indicator. This media was supplemented with 10% stripped FCS to remove any steroids in the serum. Cells were cultured in the absence or presence of 17β-estradiol (E2), an ERα and ERβ agonist; the highly selective ERα antagonist 1,3-Bis(4-hydroxyphenyl)-4-methyl-5-[4-(2-piperidinylethoxy)phenol]-1H–pyrazole dihydrochloride (MPP), or ERβ antagonist 4-[2-Phenyl-5,7-bis (trifluoromethyl) pyrazolo[1,5-a]pyrimidin-3-yl]phenol (PHTPP) (Tocris Bioscience, Bristol, UK). The 5′-bromo-2′-deoxyuridine (BrdU) cell proliferation assay kit (Roche-Applied-Science, Burgess Hill, UK) was used to measure replication of genomic DNA as an indirect parameter of the cell proliferation rate. The Caspase-Glo 3/7 apoptosis assay (Promega, Southampton, UK) and the lactate dehydrogenase activity (LDH) assay (Sigma-Aldrich, Poole, UK) were used to determine the cell proliferation rates in the presence of the MPP or PHTPP.

PNT1a cells were obtained from M. Littmann (Baylor College of Medicine, Houston). The human prostate cancer cell line, LNCaP was obtained from American Type Culture Collection (ATCC). PC3-M cells were obtained from R. C. Bergan (Northwestern University, Chicago). 3β-androstane-diol (3β-Adiol) and PHTPP experiments were performed by incubating cells with 3β-Adiol (5 μM; Sigma) or PHTPP (10 μM; Tocris) for 2–3 days. The generation of ERβ-ablated PNT1a cells, PHD2-ablated PNT1a cells and HIF-1α ablated cells has been described previously [9 (link)]. IKKβ ablated PC3-M cells were generated using shRNAs (Open Biosystems; TRCN0000018918 and TRCN0000018919). Stable cell transfectants were generated by puromycin or hygromycin selection (0.5 μg/mL for PNT1a and 2 μg/mL for PC3-M cells). The resultant ERβ, HIF-1α-ablated cells were used for subsequent experiments. For experiments involving hypoxia, cells were incubated with 100 μM cobalt chloride for 22–24 hours.