After 72 hours, cells were rinsed with PBS and placed in treatment medium composed of DMEM/F12 supplemented with 5% dextran charcoal stripped fetal bovine serum (DCC, Gemini Bio-products, Sacramento, CA, US, no. 100-119), nonessential amino acids, 6ng/mL bovine insulin and gentamicin for 48 hours. Cells were then exposed to 0.01 nM, 0.1 nM or 1nM 17β estradiol (E2, SigmaAldrich, St. Louis, MO, USA, no. E8875), 5 nM propyl pyrazole triol (PPT, Tocris, Minneapolis, MN, USA, no 1426) or vehicle control (dimethylsulfoxide , DMSO, Sigma Aldrich, no. D8418) in fresh treatment medium for 4 or 8 hours.
Growth protocol
MCF-7 cells were seeded at a density of 300,000 cells/well in 6-well plates and allowed to grow for 72 hours in complete growth medium composed of DMEM/F12 media supplemented with 10% fetal bovine serum (FBS, Atlanta Biologicals, Flowery Branch, GA), non-essential amino acids, 10µg/mL bovine insulin and gentamicin.
Extracted molecule
total RNA
Extraction protocol
Total RNA from MCF-7 cells was extracted using TRIzol Reagent (Sigma Aldrich, no. T9424) according to manufacturer’s instruction, and purified using RNeasy Mini Kit (Qiagen). Purified RNA was quantified by using NanoDrop ND-1000 spectrophotometer and the quality of RNA was analyzed by using Agilent Bioanalyzer (Agilent).
Label
Cy3
Label protocol
cRNA probes were synthesized and Cy3 labeled using Agilent LowInput QuickAmp Labeling Kit (Agilent) from 100 ng of total RNA. After purification, RNA probe concentration and dye incorporation were measured using NanoDrop ND-1000 spectrophotometer.
Hybridization protocol
Probes were hybridized to Agilent SurePrint G3 human whole genome 8x60K microarray (Agilent) following manufacture’s protocol.
Scan protocol
Microarrays were scanned with an Agilent microarray scanner.
Description
Gene expression in MCF-7, exposed to E2, 1nM, 8h
Data processing
Images were processed using Feature Extraction software, version 11.5.1.1 (Agilent).
