Supplemental Figure 1: ERa expression in MCF7 cells' MCF7 cells were either pretreated with ICI 182,

1
Lane 1 2 3 4 5 6
7 8
Supplemental Figure 1 ERa expression in MCF-7
cells. MCF-7 cells were either pre-treated with
ICI 182,780 (lanes 5-8) or EtOH (vehicle, lanes
1-4) for 6 h and then treated with EtOH, 10nM E2,
100nM 4-OHT, or the combination of E2 and 4-OHT
for 24 h. prior to preparation of WCE. 40 ug WCE
protein were separated on a 10 SDS-PAGE gel, and
immunoblotted for ERa with AER320 (Neomarkers).
The membrane was stripped and reprobed for
b-actin. The resulting film was scanned and
quantified using UnScanIt. The ERa/b-actin ratio
was determined and compared to EtOH which was set
to 100 (lane 1).
2
1.5
T47D
1.0
Fold change
0.5
0.0
EtOH
E2
Supplemental Figure 2 E2 does not regulate
miR-21 in T47D cells. T47D breast cancer cells
were treated with EtOH (vehicle) or 10 nM E2 for
6 h. Q-PCR data on (mature) miR-21 expression are
fold increase compared to EtOH and were
calculated as described in Materials and Methods.
Values are the average of 3 separate experiments
SEM.
3
7.0
MCF-7 Q-PCR
ERa
ERb
6.0
5.0
4.0
Fold change
3.0
2.0
1.0
0.0
EtOH
E2
PPT
DPN
EtOH
E2
PPT
DPN
siControl
siERa
Supplemental Figure 3 siRNA ERa knockdown in
MCF-7 cells. MCF-7 cells were transfected with
siControl or siERa for 48 h as described in
Materials and Methods. Cells were then treated
with EtOH or 10 nM E2, PPT, or DPN for 6 h. RNA
was harvested and Q-PCR performed using Taqman
primer/probe sets from ABI as described in
Materials and Methods. Shown are the mean SEM
for triplicate determinations from a
representative experiment.
4
siERa
siControl
EtOH
EtOH
DPN
PPT
DPN
PPT
Ab
E2
E2
ERa
A
HC-20
ERa
B
AER320
1.2
C
HC-20
AER320
siControl
1.0
0.8
Relative ERa
0.6
siERa
0.4
0.2
0.0
EtOH
E2
PPT
DPN
EtOH
E2
PPT
DPN
Supplemental Figure 4 siRNA ERa knockdown in
MCF-7 cells. MCF-7 cells were transfected with
siControl or siERa for 48 h as described in
Materials and Methods. Cells were then treated
with EtOH or 10 nM E2, PPT, or DPN for 24 hr. 10
ug WCE protein was applied to each well of a
slot-blot apparatus. Shown are western slot
blots for ERa using ERa antibodies HC-20 from
SantaCruz (A) or AER320 from Neomarkers (B).
Quantitation of the signal of ERa is shown in C.
5
A
B
1.2
ERa
1.0
ERb
0.8
0.6
Fold change
0.4
0.2
0.3
0.0
EtOH
E2
EtOH
E2
0.2
siControl
siERb
ERb/b-actin
C
2.5
0.1
PDCD4
PTEN
BCL2
2.0
0.0
10
20
30
20
30 nmoles
10
0
siControl
siERb
1.5
Fold change
1.0
0.5
0.0
EtOH
PPT
DPN
EtOH
E2
PPT
DPN
E2
siControl
siERb
Supplemental Figure 5 siRNA ERb knockdown in
MCF-7 cells. MCF-7 cells were transfected with
siControl or siERb for 48 h and then treated with
EtOH or 10 nM E2 for 6 h as described in
Materials and Methods. A) Q-PCR for ERa and ERb
mRNA expression reveals a 70 knockdown of ERb
mRNA. B) WCE were prepared and 40 mg of protein
were separated on a 10 SDS-PAGE gel. Western
blot was performed with H150 ERb antibody. The
membrane was stripped and reprobed for b-actin.
The ERb/b-actin ratio is plotted. 30 nmoles
siERb resulted in a 64 knockdown of ERb
protein. C) Q-PCR for PDCD4, PTEN, and BCL2 in
MCF-7 cells transfected with siControl or siERb
(48 h) and treated with 10 nM E2, PPT, or DPN for
6 h. Values are the average of 4 separate
determinations SEM.
6
A
B
Supplemental Figure 6 E2-ERa binding in MCF-7
cells overlaps miR-21. The data used for the
figure were derived from http//research4.dfci.har
vard.edu/brownlab//datasets/ER_MCF7_whole_human_ge
n ome/Hg18/ERFDR20_hg18.bed from Myles Browns
online database of genomic E2-ERa binding sites
in MCF-7 cells using the UCSC Genome browser
http//genome.ucsc.edu/cgi-bin/hgGateway for the
human March 2006 assembly. Also shown is the
location of TMEM49 and the high conservation of
the miR-21 site. A) Chr 17 55,269,845-55,277,044
both E2-ERa (black) binding overlaps with the
71 bp miR-21 gene (MIRN21, blue). B) Chr 17
55,273,408-55,273,479 E2-ERa binding (black)
overlaps with the 71 bp miR-21 gene (blue).
Special thanks to Drs. Myles Brown and Mathieu
Lupien from Harvard and Dr. Ted Kalbflesich
(UofL) for their help with these data.