|
| Status |
Public on Sep 02, 2014 |
| Title |
PD-DMEM-2 |
| Sample type |
RNA |
| |
|
| Source name |
Human Heptoma (HepG2)
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HepG2
treatment: 2.5M PD98059-complete medium
|
| Treatment protocol |
The MEK inhibitor PD98059 (Sigma-Aldrich, St. Louis, MO) was diluted in dimethyl sulfoxide (DMSO). Cells were pre-treated with an equal volume of DMSO (control) or PD98059 for 1 h prior to activation of the AAR for the indicated times in the continued presence of inhibitor
|
| Growth protocol |
All of the cell lines used in these studies were cultured in Dulbecco’s modified Eagle’s medium (DMEM, pH 7.4, Mediatech, Herndon, VA), supplemented with 1X non-essential AA, 2 mM glutamine, 100 μg/ml streptomycin sulfate, 100 U/ml penicillin G, 0.25 μg/ml amphotericin B, and 10% v/v fetal bovine serum.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the Qiagen RNeasy kit (Qiagen), including DNase I treatment before the final elution to eliminate any DNA contamination, following the manufacturer’s directions.
|
| Label |
biotin
|
| Label protocol |
A 100 ng aliquot of total RNA from four independent incubations for each of four conditions (DMEM vs DMEM lacking histidine, each with or without 2.5 uM PD98059) was amplified using the GeneChip® WT PLUS Reagent Kit (Affymetrix, Santa Clara, CA) following the manufacturer’s instructions and then 5.5 ug of cDNA was fragmented and terminally labeled.
|
| |
|
| Hybridization protocol |
Labeled targets were hybridized to Affymetrix GeneChip® Human Transcriptome Array 2.0 (HTA 2.0) for 16 h at 45°C and washed according to Affymetrix standard protocols.
|
| Scan protocol |
The Genechips were scanned with an Affymetrix G7 scanner.
|
| Description |
2.5M PD98059-complete medium
|
| Data processing |
For gene expression analysis, arrays were normalized using RMA as implemented in Partek Genomics Suite 6.6 (Partek Incorporated, St Louis MO).
probe group file:HTA-2_0.r1.pgf
meta-probeset_file: HTA-2_0.na33.1.hg19.transcript.csv
|
| |
|
| Submission date |
Jun 26, 2014 |
| Last update date |
Sep 02, 2014 |
| Contact name |
Michael Kilberg |
| E-mail(s) |
mkilberg@ufl.edu
|
| Organization name |
University of Florida
|
| Department |
Biochemistry & Molecular Biology
|
| Lab |
Kilberg
|
| Street address |
1600 SW Archer Road
|
| City |
Gainesville |
| State/province |
Florida |
| ZIP/Postal code |
32610 |
| Country |
USA |
| |
|
| Platform ID |
GPL17586 |
| Series (1) |
| GSE58869 |
HepG2-MEK Inhibition with PD98059 |
|
