MethodologyU937 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 chipsScanned 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

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_ID Gene Symbol Gene Name Control E2 5µM 4NP 5µM147628 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

RT-PCR Gene Profile of ESR2

020406080

100120140

Control (NoTreatment)

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

Treatments

mRN

A/B

-act

in m

RNA

(%)

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

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

Gene Expression Profile of BRCA1

00.10.20.30.40.50.60.70.80.911.1

1.21.3

No Treatment(Control

E2 (5µM) 4-NP (5µM)

Treatments

Rel

ativ

e G

ene

Expr

essi

on(s

igna

l of t

reat

men

t chi

p/sig

nal o

fco

ntro

l chi

p)

Rel

ativ

e G

ene

Exp

ress

ion

(Sig

nal o

f tre

atm

ent c

hip/

sign

al o

f chi

p tre

atm

ent)

Gene Expression Profile of BRCA2

00.10.20.30.40.50.60.70.80.91

1.11.21.3

No T reatment(Control

E2 (5µM) 4-NP (5µM)

Treatments

Rel

ativ

e G

ene

Expr

essi

on(s

igna

l of t

reat

men

t chi

p/sig

nal o

fco

ntro

l chi

p)

Rel

ativ

e G

ene

Exp

ress

ion

(Sig

nal o

f tre

atm

ent c

hip/

sign

al o

f chi

p tre

atm

ent)

# Of Genes Induced By E2 and4-NP

by a > or = 2-Fold Change from theControl

E2 4-NP

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)

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 MapB

Graphical Representation of Significantly Altered Genes

Signal-to-Noise Values of Four Genes Involved in Estrogen Signaling