cell line: HepaRG cell type: Cell culture time of exposure: 24 hours agent: 2,4-D dose (um): 3
Treatment protocol
Cells were exposed in 96-well plates. 2,4-dichlorophenoxyacetic acid, fenbuconazole, and imazalil were purchased from Sigma-Aldrich (St. Louis, MO). Stock solutions of 2,4-D, fenbuconazole, and imazilil were diluted in DMSO and added to culture media to a final DMSO concentration of 0.2%. The cells were exposed for 24hrs to the DMSO vehicle (0.2%) or one of the following chemical concentrations: 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, and 10uM.
Growth protocol
HepaRG cells and media were purchased from Thermo Fisher Scientific (Waltham, MA). HepaRG cells thawed and plated according to the manufacturer’s protocol onto a collagen type 1 coated (2.5ug/well) 96 well plates (day 0) and allowed to attach for 24hrs at 37C in a humidified chamber with 95% relative humidity and 5% CO2. On Day 1, 3, and 5, the media was removed from the wells and replaced with 100uL/well of maintenance/metabolism supplemented media. On day 7, the media was removed and replaced with 100uL/well of induction supplemented media. On day 8, the cells were treated with chemical.
Extracted molecule
total RNA
Extraction protocol
Trizol® (ThermalFisher) organic extraction with Phase Lock Gel™ -Heavy- tubes (5Prime). Aqueous phase placed in RB(QiaCube sample) tubes and loaded into QiaCube, the RNeasy Mini Protocol used.
Label
biotin
Label protocol
Affymetrix, GeneChip® High Throughput 3' IVT PLUS Reagent Kit
Hybridization protocol
Affymetrix, GeneChip® High Throughput 3' IVT PLUS Reagent Kit, page40-42 and GeneTitan® Instrument User Guide for Expression Array Plates
Scan protocol
Arrays were scanned on an Affymetrix GeneTitan array scanner to generate CEL files with corresponding intensity values.
Description
Gene Expression data from HepaRG cell culture BW_3_24h_3_P1_2-4-D-HepaRG
Data processing
Affymetrix CEL files were normalized by RMA using Partek Genomic's Suite 6.6 (each group normalized independently)
