S3; Pignataro et al. 2007). Database search For all genes analyzed, mouse genomic DNA sequences were obtained from the National Center for Biotechnology Information (NCBI; National Institutes of Health) and Mouse Genomic Informatics (Jackson Laboratory, Bar Harbor, ME) databases. DNA sequence analyses were performed using the Vector NTI program (Invitrogen). Candidate genes were Inhibitors,research,lifescience,medical designated as those containing one of more ARE core motifs,

CTGNGTC, and at least eight matches of the 11 bp sequence of the complete ARE (TCTGCGTCTCT) anywhere between 0.5 kb upstream of the ATG or downstream in exon/intron region. Statistical analysis Details of the statistical analysis and P values of the data are included in the figure legends, as appropriate. All data are presented as mean ± SEM. In all cases Inhibitors,research,lifescience,medical in which inmmunoblots or images are shown, the data are representative of at least three experiments with similar results. Supplemental data Supplemental data are available as Supporting Information. Results Genome profiling of ARGs In this study, we used primary cultures containing a relatively pure (≥90%) population of cortical mouse astrocytes to investigate the effects of a brief alcohol exposure on gene expression. Gene expression data were

generated from ethanol-treated (60 mmol/L, 1 h) primary cultures that were >90% positive for the mature astrocytic Inhibitors,research,lifescience,medical marker GFAP. Immunocytochemical analysis of the cultures for the microglial markers coronin-1a and isolectin-B4 (Calka et al. 1996; Chung and Han 2003; Yokoyama et al. 2004;

Yenari et al. 2006; Ahmed et al. 2007) revealed that the microglial contamination is less than ~3% (Fig. S1). The ethanol concentration used in this study (60 mmol/L), although relatively high, is well within the Inhibitors,research,lifescience,medical range associated with human intoxication, as chronic alcoholics regularly sustain blood alcohol concentration levels between 50 and 100 mmol/L and often function normally when Inhibitors,research,lifescience,medical their levels exceed 100 mmol/L (Urso et al. 1981). Gene array analysis Two thousand and four hundred transcripts were OSI-906 in vitro identified as differentially expressed across the treatment groups (using an adjusted ANOVA test at a corrected P level of ≤0.05). There was a substantial overlap of ~85% between genes significantly expressed in response to enough heat shock or ethanol treatment, suggesting that the transcriptional response to ethanol resembles the heat shock response. We have reported similar findings in our previous work in neurons (Pignataro et al. 2007; Varodayan et al. 2011). Indeed, unsupervised hierarchical cluster analysis on genes and conditions clearly shows the high degree of similarity in gene expression patterns between the ethanol and heat treatments. The ethanol-treated samples cluster was distinct from the data for the heat shock samples, however, suggesting there are also some important differences between the glial responses to heat and ethanol (Fig. 1A).