Tc s 7009

MDA-MB-231, MCF7, and MCF10A cells were obtained from the from the American Type Culture Collection and maintained as suggested. These were tested for mycoplasma contamination and characterized by short tandem repeat and Q-band assays. Normoxic cells were maintained in a humidified chamber (ambient O₂, 5% CO₂, and 37 °C). Hypoxic cells were incubated in a HypOxystation H35 (HypOxygen) at 1% O₂, 5% CO₂, and 37 °C. The hypoxia mimetic DMOG (Cayman Chemical) was used at 1 mM for 0.5, 1, and 2 h in normoxia. The HIF-1α inhibitor Echinomycin (Tocris; 20 nM) and the HIF-2α inhibitor TC-S 7009 (Tocris; 100 μM) were given to cells for 24 h before hypoxic incubation. Echinomycin is a DNA-binding agent that specifically blocks HIF-1 DNA binding activity (51 (link)). TC-S 7009 binds the HIF-2α PAS-B domain to disrupt its heterodimerization with HIF-1β and decreasing HIF-2 DNA binding (Tocris). The specific inhibitor of IGF1R autophosphorylation, AG1024 (MedChemexpress; 10 μM), was given to cells 1 h prior to treatment with NPYR agonists (Tocris; 10 nM): NPY (cat#1153), NPY1R specific (Cat#1176), or NPY5R specific (cat#1365).

BeWo cells were cultured for 24 h. Then, the medium was replaced with fresh medium containing 0.1% DMSO as a vehicle control or 30 μM TC-S7009 (Tocris Biosciences, Bristol, UK). After incubation for 24 h under chemically mimicked hypoxic conditions, the cells were subjected to cell viability assays or qRT-PCR analysis.