TGF-beta and TLR4-induced transcriptional response

To assess the pattern of TGF-beta and TLR4-induced gene expression changes at the genome-wide level, confluent healthy skin fibroblasts were incubated with the classic TLR4 ligand, LPS, TGF-beta or transfected with CD4TLR4. At the end of incubation, total RNA was harvested and processed for hybridizations to Illumina Human HT-12V4 microarray chips. Part 1: Seed three FS fibroblasts (passage 4) in DMEM (10% FBS) in 12-well or 100mm dishes at confluence, change to fresh serum-free DMEM medium with 0.1% BSA for 24 hours. Triplicate treatment: Control---no treatment, LPS (1ug/ml) for 24h, TGF-beta (10 ng/ml) for 24h, and LPS and TGF-beta for 24h. Part 2. Foreskin fibroblasts transfected and after five hrs media changed to 0.1% BSA for 48h. Then empty vector transfected (8ug/100mm dish), CD4TLR4 transfected (8ug/100mm dish), or LPS (1ug/ml) for 6h.