Quiescent cells were washed with K+-free DMEM (Sp-DMEM; Invitrogen) and incubated for 6 hr at 37oC in a humidified atmosphere with 5% CO2/balance air in control DMEM, K+-free DMEM and DMEM containing 3 mM ouabain.
Growth protocol
Rat aortic smooth muscle cells (RASMC), purchased from Lonza (Walkersville, MD, USA), were grown at 37oC in a CO2 incubator in Dulbecco’s modified Eagle medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 g/ml streptomycin, and subjected to less than 10 passages. To establish quiescence, the cells were incubated for 24 hr in media in which FBS concentration was reduced to 0.2%.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from cells grown in 6-well plates with TRIzol® reagent (Invitrogen) and purified with RNeasy® MinElute cleanup kit (Qiagen, Valencia, CA, USA), following the manufacturers’ protocols.
Label
biotin
Label protocol
The Fragmented Single-Stranded DNA was labeled by TdT with the Affymetrix® DNA Labeling Reagent that is covalently linked to biotin.
Hybridization protocol
2.5 µg of fragmented and labeled ss-DNA were hybridized onto the chip for 16 hr at 45°C. The chips were washed and stained in the GeneChip® Fluidics Station 450, using the fluidic protocol FS450_0007.
Scan protocol
Rat Gene 1.0 ST arrays were scanned using the GeneChip® Scanner 3000 7G. The software used for image processing was AGCC version 3.2.
Description
gene expression data from aortic smooth muscle cells
Data processing
Partek Genomics Suite (Partek, St. Louis, Missouri) was used for data analysis. The data were normalized by Robust Multichip Average (RMA) algorithm with adjustment for GC content, which uses background adjustment, quantile normalization and summarization.
