1. Methodology U937 Human Immune Cells Control (No treatment) (n=4) Estrogen (5 uM) (n=4) 4-nonylphenol (5 uM) (n=4) Cultured Cells, RNA Isolation, RT (to cDNA) Applied cDNA to microarray chips Scanned chips for detection of gene expression by chemiluminescence Data analyzed by Spotfire software RT-PCR (using ER-beta) Reference gene: B-actin 2% agarose gel electrophoresis 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 A B Genes altered with E2 treatment after 48 hours Genes altered with 4-NP treatment after 48 hours 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. Probe_IDGene SymbolGene NameControlE2 5µM4NP 5µM 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 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 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. Gene profiles of BRCA1 and BRCA2. The E2 treament induced a fold repression of more than two fold while the NP treatment had no significant effect. This phenomenon was observed for 23/45 genes in the portfolio involved in the onset of breast cancer. Gene Expressions Profiles of BRCA1 and BRCA2 Relative Gene Expression (Signal of treatment chip/signal of chip treatment) Relative Gene Expression (Signal of treatment chip/signal of chip treatment) # Of Genes Induced By E2 and 4-NP by a > or = 2-Fold Change from the Control E24-NP 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) A (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. Comparison of Induced Genes/Hierarchical Clustering Heat Map About 45.0% (14,913) exhibited significant changes in gene expression (> 2-fold) by E2 while approximately 1.91% (633) B E2 (5uM) 4-NP (5uM) Hierarchical Clustering Normalized Heat Map B Graphical Representation of Significantly Altered Genes Signal-to-Noise Values of Four Genes Involved in Estrogen Signaling