Microarray analysis of LPS responses of thioglycolate-elicited macrophages and counter-regulation by glucocorticoid receptor, PPARγ and LXR-specific ligands
Experimental design
Thioglycollate-elicited macrophages were isolated from wild type or IRF3-deficient mice by peritoneal lavage 3 days following peritoneal injection of 2.5 ml 3% thioglycollate (DIFCO). Cells were plated in RPMI medium 1640 and 10% fetal bovine serum, washed after 5 h the medium was removed and cells were fed with fresh medium containing 0.5% fetal bovine serum. Cells were treated with LPS (Sigma) a concentration of 100 ng/ml or poly I:C (100 ng/ml) in the absence or presence of receptor-specific agonists for the glucocorticoid receptor (dexamethasone), PPARγ (GW7845) or LXR (GW3965) at 1 µM concentrations. Cells were lysed with Trizol (Invitrogen) six hours following LPS or poly I:C treatment and total RNA was purified using RNeasy columns (Qiagen). cRNA was generated from 10 ug total RNA using Superscript (Invitrogen) and the High Yield RNA transcription labeling kit (Enzo). Fragmented cRNA was hybridized to Codelink mouse Uniset 1 microarrays according to manufacture's instructions. Data was analyzed with Microarray Suite (Affymetrix), GeneSpring (Silicongenetics) and in-house software developed as described (Ogawa et al., 2004). Two to four biological replicates were performed for each experimental condition.Reference: Molecular Determinants of Crosstalk between Nuclear Receptors and Toll-like Receptors Mediating Counter-regulation of Inflammatory Responses. Sumito Ogawa, Jean Lozach, Chris Benner, Gabriel Pascual, Rajendra K. Tangirala, Stefan Westin, Alexander Hoffmann, Shankar Subramaniam, Michael David, Michael G. Rosenfeld, and Christopher K. Glass. Cell, Vol 122, 707-721, 9 September 2005.
Query Interface
GOby-rendered report pages.
Click "Term table" to access annotation terms that are enriched among differentially expressed features. Click "Tree View" to display the hierarchical structure of the annotation system with corresponding enrichment likelihoods.| WT NOTX vs KO+LPS | Term table | Tree View (1.8M) | |
|---|---|---|---|
| WT NOTX vs KO NOTX | Term table | Tree View (616k) | |
| WT NOTX vs WT+LPS | Term table | Tree View (1.4M) | |
| WT NOTX vs WT+LPS+DEX | Term table | Tree View (1.3M) | |
| WT NOTX vs WT+LPS+LXR | Term table | Tree View (1.7M) | |
| WT NOTX vs WT+LPS+PPARg | Term table | Tree View (1.5M) | |
| WT NOTX vs WT+PIPC | Term table | Tree View (1.4M) | |
| WT NOTX vs WT+PIPC+DEX | Term table | Tree View (1.5M) | |
| WT NOTX vs WT+PIPC+LXR | Term table | Tree View (1.6M) | |
| WT NOTX vs WT+PIPC+PPARg | Term table | Tree View (2.0M) |
GOby is a gene annotation tool that identifies statistically overrepresented gene groups, and is available as part of the VAMPIRE Microarray analysis suite.
Reference: VAMPIRE microarray suite: a web-based platform for the interpretation of gene expression data Albert Hsiao, Trey Ideker, Jerrold M. Olefsky, and Shankar Subramaniam. Nucleic Acids Research, Nucleic Acids Research 2005 33(Web Server issue):W627-W632; doi:10.1093/nar/gki443
Download Normalized Data
Explanation of column headers in *normalized data
WT stands for "Wild-type" and KO stand for the "IRF3 (-/-) knock out mice"NOTX - no treatment (Control)
LPS - treatment with lipopolysaccharide (binds to TLR4)
PIPC - treatment with PIPC (binds TLR3)
DEX, LXR, and PPARγ are always paired with either LPS or PIPC in these experiments since we are studying their effect on inflammation.
DEX - treatment with dexamethasone (binds to GR)
LXR - treatment with compound GW3965 (binds to LXR)
PPAR/PPARG - treatment with compound GW7845 (binds to PPARγ)
All experiments were carried out 6 hours after being treated.
*Reference: Statistical analysis of high-density oligonucleotide arrays: a multiplicative noise model. Sasik et al. Bioinformatics. 2002 Dec;18(12):1633-40
