Transcript (5 uM) (n=4)

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
Spotfire Graphical Representation
of Significantly Altered Genes
Comparison of Altered Genes (By at
least 2-Fold from Control)
B
A
E1
4-NP
Estrogen
All genes
from the
wholegenome
nanochips
(15,960/33,155)
Total Genes altered by 4-NP
14,913
414
633
(43.4%)
(2.6%)
(4.0%)
Total Genes altered by estrogen
C
D
E2
4-NP
Estrogen
Genes
from
customized
portfolio
(69/90)
Significantly altered genes by 4-NP
54
7
8
(78.3%)
(10.1%)
(11.6%)
Significantly altered genes by estrogen
A/B) Scatterplots that represent Total gene expression analyzed by DNA microarray. The gene ID is represented
by the x-axis and the fold change in gene expression are represented by the y-axis. (C/D) Scatterplots represent
Only the significantly altered genes exhibiting at least a 2-fold-change from control. The x-axis represents the
gene ID and the y-axis represents fold change in gene expression.
(E1) Venn Diagram shows the numerical comparison of the total genes altered by 4-NP and estrogen as
depicted in Figure A/B. (E2) Venn Diagram shows numerical representations of the gene expression of
only the 90 portfolio genes. Portfolio genes include a total of 90 genes involved in estrogen signaling and
breast cancer initiation.
Signal-to-Noise Values of Four Genes
Involved in Estrogen Signaling
F1
F2
Probe_ID
ERα
ERβ
Gene Symbol
Gene Name
Control
E2 5µM
4NP 5µM
147628 ERα
estrogen receptor alpha
3.39
-0.56
5.23
202485 ERβ
estrogen receptor beta
13.22
2.34
1.87
171023 ERRβ
estrogen-related receptor beta
46.7
32.35
29.92
(F1) 4-NP and estrogen have the potential to down regulate the estrogen receptor beta (ERβ) and the estrogen receptor
related beta (ESRR-β) and up regulate repressor estrogen receptor activity (REA). ERα does not exhibit a significant
pattern.
(F2) Chart depicting numerical values for up/down regulation of gene after treatment. ERβ gene was used towards RTPCR validation step.
ERRβ
RT-PCR Gene Expression Profile
Statistical Significance
β-actin
ERβ
RT-PCR Gene Profile of ERβ
G2
(G1) ERβ gene expression.
Lane 1=control, Lane 2 =E2
5uM, Lane 3=control and Lane
4 = 4-NP 5uM.
(G2) β-actin reference gene
expression.
Lane 1=control, Lane 2 =E2
5uM, Lane 3=control and Lane
4 = 4-NP 5uM.
1
2
3
4
1
2
3
4
mRNA/B-actin mRNA (%)
G1
G3
140
120
100
80
60
40
20
0
n=4
n=4
n=4
Control (No
Treatment)
E2 (5 uM)
Treatments
4-NP (5 uM)
(G3) Graphical representation of RTPCR Analysis of Intensity of ERβ gene
expression normalized with the internal
control gene β-actin. This step serves
as a validation for the microarray data.