1. Methodology Control (no treatment) Estrogen (5 uM) 4-nonylphenol (5 uM) Cultured Cells, Isolated RNA, RTed to cDNA Data analyzed by Spotfire software RT-PCR performed with ESR2 gene and beta-actin reference gene Applied cDNA to microarray chips. Which were then scanned for detection of gene expression by chemiluminescence U937 Human Immune Cells (ATTC, USA) Scanned chip containing DNA oligo information. A display of the gene profiling capabilities offered by microarray technology Figure 2: (A) Scatterplots of gene expression analyzed by DNA microarray. The probe ID’s for each gene is represented by the x-axis and the fold changes in gene expression for the experimental samples based on the fluorescence-based detection signals of the mRNA levels in the control are represented by the y-axis. (B) Remaining genes after filtering by modulation and normalization with the b-actin reference gene. Genes that exhibited 2-fold regulation AB Genes altered with E2 treatment after 48 hours Genes altered with 4-NP treatment after 48 hours Gene Ontology Database Gene_ID Gene_NameGene_Symbol hCG21449.3;hCG21449.3estrogen receptor 2 (ER beta)ESR2 hCG2016877;hCG2016877peroxiredoxin 5;estrogen-related receptor alpha ESRRA;PRDX 5 hCG31941.3;hCG31941.3estrogen receptor bining proteinERBP hCG1796030.1;hCG1796030.1 peroxisome proliferative activated receptor, gamma, coactivator 1, beta PPARGC1B hCG28493.2;hCG28493.2retinol dehydrogenase 8 (all-trans)RDH8 hCG1811630.1;hCG1811630.1estrogen receptor 1ESR1 hCG20789.4;hCG20789.4egl nine homolog 2 (C. elegans)EGLN2 hCG21644.4;hCG21644.4;hCG28380.2estrogen-related receptor betaESRRB hCG2015551;hCG2015551DEAD (Asp-Glu-Ala-Asp) box polypeptide 54DDX54 hCG21644.4;hCG28380.2;hCG21644.4estrogen-related receptor betaESRRB hCG24417.4;hCG24417.4RAS-like, estrogen-regulated, growth inhibitorRERG hCG1781443.2;hCG1818318.1;hCG1781443.2UDP glycosyltransferase 2 family, polypeptide B4UGT2B4 hCG1776094.1;hCG1776094.1tripartite motif-containing 47TRIM47 hCG25939.2;hCG25939.2repressor of estrogen receptor activityREA hCG2028796.1;hCG2028796.1breast cancer anti-estrogen resistance 3BCAR3 hCG2010626;hCG2010626RNA binding motif protein 9RBM9 hCG20144.4;hCG20144.4RNA binding motif protein 14RBM14 hCG24804.3;hCG24804.3estrogen-related receptor gammaESRRG hCG1781443.2;hCG1818318.1;hCG1781443.2UDP glycosyltransferase 2 family, polypeptide B4UGT2B4 hCG1796030.1;hCG1796030.1 peroxisome proliferative activated receptor, gamma, coactivator 1, beta PPARGC1B hCG38316.2;hCG38316.2 sulfotransferase family 1E, estrogen-preferring, member 1 SULT1E1 # Of Genes Induced By E2 and 4-NP by a > or = 2-Fold Change from the Control E24-NP Hierarchical Clustering Normalized Heat Map 14,913 (45.0%) 414 (1.2%) 54 (60.0%) 7 (7.8%) 8 (8.9%) 633 (1.9%) E24-NP All genes from the whole- genome nanochips (33,155) Genes from customized portfolio (90) BA Figure 3: (A) Venn Diagram which shows the number of genes that exhibited significant changes in gene expression after the application of E2 and concentrations of NP. (B) A normalized hierarchical clustering heat map performed in order to similarities of genes and to what extent they were affected. Genes that were downregulated significantly are illustrated by the green while red denotes genes that were upregulated by at least 2-fold. E2 (5uM) 4-NP (5uM) Signal-to-Noise Values of Four Genes Involved in Estrogen Signaling Figure 5: This figure indicates that E2 and NP have the potential to down regulate the estrogen receptor beta (ESR1) and the estrogen receptor related beta (ESRR-β) and up regulate repressor estrogen receptor activity (REA) (at their respective concentrations). ESR1 does not exhibit a significant pattern. These data appear to demonstrate that NP as well as enhanced estrogen levels diminishes estrogen beta activity negatively. Signal to Noise Values For Four genes From the Portfolio/List Probe_IDGene SymbolGene NameControl E2 5 µ M4NP 5 µ M 147628ESR1estrogen receptor 13.39-0.565.23 202485ESR2estrogen receptor 2 (ER beta)13.222.341.87 171023ESRRBestrogen-related receptor beta46.732.3529.92 177753REA repressor of estrogen receptor activity38.5760.3861.04 Signal Responses from Scanned Chips Graphical Representation of Significantly Altered Genes Sample of the 90 Customized Portfolio of Genes (GeneEntrez, GeneOntology) Figure 7: RT-PCR Analysis of Intensity of ESR2 gene expression normalized with the internal control gene beta-actin. This data serves as a validation for the microarray analysis. RT-PCR Gene Expression Profile ESR2 β-actin 1 2 3 4 Figure 6: These gels exhibit expression of the ESR2 gene the β-actin reference gene. Lane 1=control, Lane 2 =E2 5uM, Lane 3=control and Lane 4 = 4-NP 5uM. n=4