cell line: CCL210 treatment: TGFb1 and PGE2 cell type: primary adult human fibroblast cell line
Treatment protocol
Cells were then treated with or without TGF-β1 (2 ng/ml, R&D Systems, Minneapolis, MN, USA) for 24 h to induce myofibroblast differentiation. Medium was the removed and cells were subsequently treated with or without PGE2 (500 nM, Cayman Chemicals, Ann Arbor, MI, USA) for an additional 48 h. This protocol generated four treatment conditions: Control, PGE2 alone, TGF-β1 alone, and TGF-β1 followed by PGE2 (Figure 1).
Growth protocol
Cells were cultured at 37° C in 5% CO2 with Dulbecco’s Modified Eagle’s Medium (DMEM, Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 100 U/ml penicillin/streptomycin (Invitrogen). At 80% confluence, cells were trypsinized, centrifuged, and plated at a density of 250,000 cells per well in 6-well plates. Cells were allowed to adhere overnight before being replaced with serum-free medium (DMEM) for 48 hours.
Extracted molecule
total RNA
Extraction protocol
RNA from independent experiments was isolated from CCL210 cells using the RNEasy kit (Qiagen, Germantown, MD, USA) per manufacturer’s instructions.
Label
biotin
Label protocol
Biotinylated aRNA were prepared according to the Affymetrix GeneChip 3' IVT Express kit protocol from 250 ng total RNA (Affymetrix GeneChip 3' IVT Express kit Instruction Manual, P/N 702646 Rev. 5)
Hybridization protocol
Following fragmentation, 7.5 ug of aRNA were hybridized for 16 hr at 45C on HG-U219 Strip Arrays using the Affymetrix Gene Atlas system (software version 1.0.4.267).
Scan protocol
Arrays were scanned using the Affymetrix Gene Atlas system.
Description
Gene expression data from fibroblasts treated with TGFb1 and PGE2
Data processing
Expression values RMA normalized via Bioconductor Affy package justRMA command
