Genes that were differentially expressed either by alcohol treatment or by phenotype were analyzed using Ingenuity Pathway Analysis (Ingenuity® Systems, Spring 2013 release). Duplicate probe sets were eliminated by selecting the entry with the best p-value. Parameters were set to use the Ingenuity knowledge base as the reference set. Due to the large number of genes that were differentially expressed after ethanol treatment, we limited the analysis to those genes with FDR ≤ 0.05 and minimum absolute fold change ≥ 1.2; for phenotype, FDR was set at ≤ 0.36 with no minimum fold change. We used the canonical pathway analysis to identify modified pathways and the upstream regulator analysis to identify putative actors responsible for the changes in expression. The upstream regulator analysis looks for transcription factors, cytokines, hormones, vitamins and other signaling molecules that may be responsible for a portion of the differential expression. IPA uses its knowledge base of causal effects and the list of differentially expressed genes to predict whether a particular regulator could be activated. The activation z-score sign (+/−) indicates whether the upstream ‘actor’ is activated or less active in either the LCLs treated with ethanol or from alcoholics.
