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Supplemental Figure 1: ER expression in MCF-7 cells. MCF-7 cells were either pre-treated with ICI...
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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). -actin ER EtO H EtO H E 2 E 2 + 4-O HT 4-O HT E 2 E 2 + 4-O HT 4-O HT IC I182,780 % ER / -actin 100 72 110 117 31 30 31 50 M CF-7 Lane 1 2 3 4 5 6 7 8
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Transcript of Supplemental Figure 1: ER expression in MCF-7 cells. MCF-7 cells were either pre-treated with ICI...
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 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 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).
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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.
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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 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.
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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 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 ER using ERantibodies HC-20 from SantaCruz (A) or AER320 from Neomarkers (B). Quantitation of the signal of ER is shown in C.
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Supplemental Figure 5: siRNA ER knockdown in MCF-7 cells. MCF-7 cells were transfected with siControl or siERfor 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 E2, PPT, or DPN for 6 h. Values are the average of 4 separate determinations ± SEM.
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Supplemental Figure 6: E2-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 E2-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 E2-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.
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