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).
Npy1r specific
Manufactured by Bio-Techne
NPY1R is a protein that functions as a receptor for the neuropeptide Y (NPY) hormone. It plays a role in regulating various physiological processes, such as food intake, energy homeostasis, and circadian rhythms. This protein is commonly used in research applications to study the mechanisms and functions of the NPY signaling pathway.
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2 protocols using npy1r specific
1
Hypoxia and HIF Inhibition in Breast Cancer
2
Hypoxic Breast Cancer Cell Response
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MDA-MB-231 and MCF7 cells were obtained from the American Type Culture Collection and maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, as suggested. They were maintained mycoplasma free in a humidified chamber (5% CO2, and 37˚C). Cells were introduced to and maintained in hypoxia by incubating them in a HypOxystation H35 workstation (HypOxygen) at 1% O2, 5% CO2, and 37˚C. Cells were treated with NPYR agonists (Tocris):1 × 10− 9 M NPY (Cat#1153), 1 × 10− 8 M NPY1R-specific (Cat#1176), or 1 × 10− 8 M NPY5R-specific (Cat#1365), and NPYR antagonists (Tocris): 1 × 10− 6 M NPY1R (BIBP 3226, Cat #2707), 1 × 10− 5 M NPY5R (L-152, Cat# 1382) For spheroid experiments, BIBP 3226 and L-152 were used at 1 × 10− 5 M and 1 × 10− 4 M, respectively. Cells were pre-incubated with antagonists for 30 min prior to agonist exposure.
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