1. Supplemental Figure 1: ER  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 E 2, 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 ER  with AER320 (Neomarkers). The membrane was stripped and reprobed for  -actin. The resulting film was scanned and quantified using UnScanIt. The ER  /  -actin ratio was determined and compared to EtOH which was set to 100 (lane 1). Lane 1 2 3 4 5 6 7 8

2. Supplemental Figure 2: E2 does not regulate miR- 21 in T47D cells. T47D breast cancer cells were treated with EtOH (vehicle) or 10 nM E 2 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. 0.0 0.5 1.0 1.5 EtOHE2E2 Fold change T47D

3. Supplemental Figure 3: siRNA ER  knockdown in MCF-7 cells. MCF-7 cells were transfected with siControl or siER  for 48 h as described in Materials and Methods. Cells were then treated with EtOH or 10 nM E 2, 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. 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 EtOHE2E2 PPTDPNEtOHE2E2 PPTDPN siControl siER  ER  ER  MCF-7 Q-PCR Fold change

4. Supplemental Figure 4: siRNA ER  knockdown in MCF-7 cells. MCF-7 cells were transfected with siControl or siER  for 48 h as described in Materials and Methods. Cells were then treated with EtOH or 10 nM E 2, 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 ER  using ER  antibodies HC-20 from SantaCruz (A) or AER320 from Neomarkers (B). Quantitation of the signal of ER  is shown in C. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 EtOHE2PPTDPNEtOHE2PPTDPN Relative ER  HC-20AER320 EtOHE2 PPT DPN EtOH E2 PPTDPN siER  siControl ER  Ab AER320 HC-20 siER  siControl A B C

5. Supplemental Figure 5: siRNA ER  knockdown in MCF-7 cells. MCF-7 cells were transfected with siControl or siER  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 ER  and ER  mRNA expression reveals a 70% knockdown of ER  mRNA. B) WCE were prepared and 40  g of protein were separated on a 10% SDS- PAGE gel. Western blot was performed with H150 ER  antibody. The membrane was stripped and reprobed for  -actin. The ER  /  -actin ratio is plotted. 30 nmoles siER  resulted in a ~64% knockdown of ER  protein. C) Q-PCR for PDCD4, PTEN, and BCL2 in MCF-7 cells transfected with siControl or siER  (48 h) and treated with 10 nM E 2, PPT, or DPN for 6 h. Values are the average of 4 separate determinations ± SEM. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 EtOHE2E2 E2E2 Fold change ER  ER  siControl siER  A B 0.0 0.1 0.2 0.3 1020302030 nmoles ER  /  -actin 100 siControl siER  0.0 0.5 1.0 1.5 2.0 2.5 EtOHPPTDPNEtOHE2E2 PPTDPN Fold change E2E2 siControl siER  C PDCD4PTENBCL2

6. Supplemental Figure 6: E 2 -ER  binding in MCF-7 cells overlaps miR-21. The data used for the figure were derived from http://research4.dfci.harvard.edu/brownlab//datasets/ER_MCF7_whole_human_gen ome/Hg18/ERFDR20_hg18.bed from Myles Brown’s online database of genomic E 2 -ER  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 E 2 -ER  (black) binding overlaps with the 71 bp miR-21 gene (MIRN21, blue). B) Chr 17: 55,273,408-55,273,479: E2-ER  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. B A