Hepatocytes were isolated and seeded at a density of 4x105 viable cells/well onto BioCoat Collagen I Cellware 12-well culture plates in William’s E Medium, supplemented with 10% FBS, 100 U/ml Pen/Strep, 2 mM L-glutamine,32 mU/ml human insulin, 1 mM sodium pyruvate, 1 x nonessential amino acids, 15 mM HEPES, and 0.8 µg/ml hydrocortisone. After 24 h fully attached cells were adapted for another 12 h to William’s E Medium, supplemented with 10% FBS, 100 U/ml Pen/Strep, 2 mM L-glutamine,32 mU/ml human insulin, 0.1 µM dexamethasone and 0.1% DMSO. Cells were treated for 24 hours in fresh medium with 1µM CITCO, 10 µM rifampicin, 50 µM WY14,643 or vehicle (0.1% dimethylsulfoxide (DMSO).
Growth protocol
After the treatment for 24h with the indicated agonists the cells were lysed.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using Rneasy kit from Qiagen according to the manufacture´s conditions. All (24) used RNA samples had an RNA integrity number (RIN) > 9 (determined with the Bioanalyzer 2100 from Agilent Technologies) to ensure high quality of RNA.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 6 ug total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on Human Gene 1.0ST Arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the GeneChip® scanner 7G (Affymetrix).
Description
Gene expression data from PHH treated with WY14,643
Data processing
After visual inspection of the obtained GeneChip images, the Affymetrix Expression Console (Affymetrix) was used for quality control of microarrays and pre-processing of expression data via robust multi-array average (RMA; Gene Level - Default).
