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.

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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 hCG ;hCG estrogen receptor 2 (ER beta)ESR2 hCG ;hCG peroxiredoxin 5;estrogen-related receptor alpha ESRRA;PRDX 5 hCG ;hCG estrogen receptor bining proteinERBP hCG ;hCG peroxisome proliferative activated receptor, gamma, coactivator 1, beta PPARGC1B hCG ;hCG retinol dehydrogenase 8 (all-trans)RDH8 hCG ;hCG estrogen receptor 1ESR1 hCG ;hCG egl nine homolog 2 (C. elegans)EGLN2 hCG ;hCG ;hCG estrogen-related receptor betaESRRB hCG ;hCG DEAD (Asp-Glu-Ala-Asp) box polypeptide 54DDX54 hCG ;hCG ;hCG estrogen-related receptor betaESRRB hCG ;hCG RAS-like, estrogen-regulated, growth inhibitorRERG hCG ;hCG ;hCG UDP glycosyltransferase 2 family, polypeptide B4UGT2B4 hCG ;hCG tripartite motif-containing 47TRIM47 hCG ;hCG repressor of estrogen receptor activityREA hCG ;hCG breast cancer anti-estrogen resistance 3BCAR3 hCG ;hCG RNA binding motif protein 9RBM9 hCG ;hCG RNA binding motif protein 14RBM14 hCG ;hCG estrogen-related receptor gammaESRRG hCG ;hCG ;hCG UDP glycosyltransferase 2 family, polypeptide B4UGT2B4 hCG ;hCG peroxisome proliferative activated receptor, gamma, coactivator 1, beta PPARGC1B hCG ;hCG 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 ESR1estrogen receptor ESR2estrogen receptor 2 (ER beta) ESRRBestrogen-related receptor beta REA repressor of estrogen receptor activity 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 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