In vivo study, aortic root, pulmonary artery, aorta and spleen were isolated from mice in 3 groups: 1) intraperitoneal injection of 20mg of LPS priming only, 2) oral administration of FK565 (100mg) for consecutive days 3) oral administration of FK565 (100mg) for consecutive days 1 day after LPS priming, at day 2, 4, and 7. Ex vivo study, each tissue of aortic root, pulmonary artery and aorta sterilely isolated from mice (BALB/c) was cultured in a 96-well plate with EBM-2 medium and EGM-2 (Lonza) in the presence of FK565 (10μg/mL, N=6) or none (N=6) in a CO2 (5%) incubator at 37°C for 24 hours.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Fibrous Tissue (Qiagen Inc., Valencia, CA, USA) and amplified using Amino Allyl MessageAmp™ II aRNA Amplification Kit (Ambion, Austin, TX, USA). RNA quality and concentration was determined by analysis with NanoDrop at the Harvard-Partners Center for Genetics and Genomics (Harvard Medical School, Cambridge, MA USA).
Label
Cy3
Label protocol
Nimblegen one-color cy3 labeling
Hybridization protocol
Hybridization was performed for 19h at 42C to the mouse Nimblegen Gene Expression arrays (090901_MM9_EXP_HX12, Roche NimbleGen, Madison, WI, USA) .
Scan protocol
GeneChips were scanned using the Gene Pix 4000B (Molecular Devices Corporation) .
Description
pulmonary artery, FK stimulation, 24hours, ex vivo
Data processing
The raw data (tif.) was subjected to RMA (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249), quantile normalization (Bolstad et al. Bioinformatics 19(2):185), and background correction as implemented in the NimbleScan software package, version 2.5. (Roche NimbleGen, Inc.).
