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

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

Gene Ontology Database Gene_IDGene_Name Gene_Symbol

hCG21449.3;hCG21449.3 estrogen receptor 2 (ER beta) ESR2

hCG2016877;hCG2016877 peroxiredoxin 5;estrogen-related receptor alphaESRRA;PRDX5

hCG31941.3;hCG31941.3 estrogen receptor bining protein ERBP

hCG1796030.1;hCG1796030.1peroxisome proliferative activated receptor, gamma, coactivator 1, beta

PPARGC1B

hCG28493.2;hCG28493.2 retinol dehydrogenase 8 (all-trans) RDH8

hCG1811630.1;hCG1811630.1 estrogen receptor 1 ESR1

hCG20789.4;hCG20789.4 egl nine homolog 2 (C. elegans) EGLN2

hCG21644.4;hCG21644.4;hCG28380.2 estrogen-related receptor beta ESRRB

hCG2015551;hCG2015551 DEAD (Asp-Glu-Ala-Asp) box polypeptide 54 DDX54

hCG21644.4;hCG28380.2;hCG21644.4 estrogen-related receptor beta ESRRB

hCG24417.4;hCG24417.4 RAS-like, estrogen-regulated, growth inhibitor RERG

hCG1781443.2;hCG1818318.1;hCG1781443.2 UDP glycosyltransferase 2 family, polypeptide B4 UGT2B4

hCG1776094.1;hCG1776094.1 tripartite motif-containing 47 TRIM47

hCG25939.2;hCG25939.2 repressor of estrogen receptor activity REA

hCG2028796.1;hCG2028796.1 breast cancer anti-estrogen resistance 3 BCAR3

hCG2010626;hCG2010626 RNA binding motif protein 9 RBM9

hCG20144.4;hCG20144.4 RNA binding motif protein 14 RBM14

hCG24804.3;hCG24804.3 estrogen-related receptor gamma ESRRG

hCG1781443.2;hCG1818318.1;hCG1781443.2 UDP glycosyltransferase 2 family, polypeptide B4 UGT2B4

hCG1796030.1;hCG1796030.1peroxisome proliferative activated receptor, gamma, coactivator 1, beta

PPARGC1B

hCG38316.2;hCG38316.2sulfotransferase family 1E, estrogen-preferring, member 1

SULT1E1

# Of Genes Induced By E2 and4-NP

by a > or = 2-Fold Change from theControl

E2 4-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%)

E2 4-NP

All genes from thewhole-genomenanochips(33,155)

Genes fromcustomizedportfolio(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_ID Gene Symbol Gene Name ControlE2

5µM 4NP 5µM

147628 ESR1 estrogen receptor 1 3.39 -0.56 5.23

202485 ESR2 estrogen receptor 2 (ER beta) 13.22 2.34 1.87

171023 ESRRB estrogen-related receptor beta 46.7 32.35 29.92

177753 REArepressor of estrogen receptor activity 38.57 60.38 61.04

Signal Responses from Scanned Chips

Graphical Representation of Significantly Altered Genes

Sample of the 90 Customized Portfolio of Genes(GeneEntrez, GeneOntology)

RT-PCR Gene Profile of ESR2

020406080

100120140

Control (NoTreatment)

E2 (5 uM) 4-NP (5 uM)

Treatments

mRNA

/B-act

in mR

NA (%

)

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 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

n=4 n=4