cDNA Microarray Analysis

Gene expression profiling was performed using an Affymetrix Gene Chip^® PrimeView™ Human Gene Expression Array, which contains 36,000 probe sets representing 20,000 well-characterized human genes (Affymetrix, Santa Clara, CA). Total RNA was isolated from duplicate cultures using the TRIzol method. After passing a quality control measurement, RNA was amplified and labeled using the Affymetrix 3ʹ IVT Express kit and hybridized to the array for 16 h. This was followed by washing and staining using the Affymetrix Fluidics Station 450/250. Arrays were scanned using an Affymetrix 3000 7G plus instrument. Affymetrix GeneChip Command Console (AGCC) software was used to generate.cel files. Data were imported into Agilent Gene Spring GX software for further analysis. Unsupervised hierarchical clustering analysis was performed using Cluster 3.0 software (Stanford University, USA). The row dendrogram was generated using Ward’s clustering method with a half square Euclidean distance measure. The column dendrogram was generated using the single linkage clustering method and a Euclidean distance measure. Gene ontology analysis was based on David 6.7 (http://david.abcc.ncifcrf.gov/home.jsp).

The transcriptome data have been deposited in the NCBI Gene Expression Omnibus and are accessible through GEO Series accession number GSE145786.