Zhou et al. BMC Genomics 2011, 12:124 http://www.biomedcentral.com/1471-2164/12/124 Page 9 of 17 Figure 4 Illustration of Gene Set Enrichment Analysis (GSEA) informatics with neurotrophic factor related gene set. (Left panel) Profile of the running enrichment score (ES) and positions of a prominent neurotrophic factor related gene set, GO:0040007: Growth, on the rank ordered list GSEA output for the comparison ALC vs. CONTROL. This test is a one-way test, i.e. whether gene expression is higher in control than in ALC. The x-axis lists all the genes ranked based on their associations with phenotype, i.e. the comparison ALC vs. CONTROL. The blue vertical bars indicate candidate genes in the target gene set. The ES profile records the cumulative score of the gene ranks from the target gene set. If a majority of gene ranks from the candidate gene set are high (i.e. toward the start of ranking) compared to the rest of genes, the cumulative ranking score (profile) will have a high peak, suggesting a significant enrichment of this gene set. The statistical significances (p-value) were calculated based on the height of this peak through a permutation test (p-value = 0.010 in Experiment 1 and 0.005 in Experiment 2). (Right panel) The significant genes (enriched in control) are determined by the position of the peak of the profile. There are 21 candidate genes up to this peak position which are claimed as significant. They are plotted in the Heatmap (green means high expression level, and red means low expression level) in Experiment 2. pluripotency and embryonic stem cells [40]), extracellular matrix (ECM) molecules, and ECM protease inhibitors (Table 5). Three gene sets were down-regulated in the ALC-NTC subgroup (i.e., enriched in the control group): other related growth factors (Neural specification), other regulators of cell differentiation, and ECM protease inhibitors (Table 5). Two gene sets were down-regulated in the ALC-NTO group (enriched in the control group): other related growth factor and other ECM molecules (Table 5). There were no significant gene sets in compari- sons between ALC-NTC and ALC-NTO. No gene set was enriched in any alcohol-treated group. Table 5 GSEA for Stem Cell Related Gene Sets Comparison Gene Set Control vs ALC Other ECM Molecules~^ ECM Protease Inhibitors~,# TGF-b Activin-responsiv~ e Other Regulators of Cell Differentiation (Neural Specification) # Other Related growth Factor^ Other Related growth Factor# ~= Gene set is reduced in ALC (all alcohol group) as compared with Control. ^= Gene set is reduced in ALC-NTO as compared with Control. #= Gene set is reduced in ALC-NTC as compared with Control. ECM: extracellular matrix. Size 9 7 16 17 7 p-value Exp. 2 0.002 0.004 0.010 0.003 0.005 0.040 Significant Genes Ctgf, Thbs2, Tgfbi, Ecm1 Thbs1, Timp3 Junb, Fos, Tgfbi, Pdgfb, Tgfb1i1, Igf1 Elavl3, Neurod1, Neurod4, Nhlh1, Neurog1, Nhlh2, Neurog3, Spock2, Neurog2 Ctgf, Igf1 Ctgf Hgf Igf1 p-value Exp. 1 0.016 0.006 0.014 0.004 0.015 0.008