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. Author manuscript; available in PMC: 2013 Jun 16.
Published in final edited form as: Endocrine. 2010 Nov 11;39(1):48–61. doi: 10.1007/s12020-010-9411-8

Figure 2.

Figure 2

Fig. 2 Synthesis of functional ERs. For Western blot (WB) (a) and Electrophoretic Mobility Shift Assay (EMSA) (b), MDA-MB-231 cells were infected with the parent recombinant adenovirus (Ad5) at MOI 600, a recombinant adenovirus bearing ERβ cDNA at 600 MOI or ERα cDNA at 100 MOI together with 500 MOI of Ad5 to equalize the total adenovirus concentration. a Infected cells were collected at indicated times, and cell extracts (10 μg) were subjected to WB. Molecular mass in KDa is indicated. b For EMSA, 10 μg extracts of infected cells for the indicated times were incubated with radiolabeled DNA fragment containing the consensus ERE sequence for 1 h in the absence or presence of a flag antibody. Reactions were subjected to 5% non-denaturing PAGE. The gel was dried and exposed to PhosphorImager. Free depicts unbound ERE. ER–ERE indicates the radiolabeled ERE-bound-ERs in the absence or presence of the antibody. c For immunocytochemistry, infected cells were probed with a fluorescein-conjugated Flag antibody (FITC) at 48 h postinfection. DAPI was used to stain nuclei. d For in situ 3H-E2 Binding Assay, cells were infected with adenoviruses in the absence E2 for 48 h. Cells were then incubated in fresh medium containing 10−7 M of 3H-E2 for 1 h. Cells were collected, and the radioactivity retained in cells was quantified by a scintillation counter. The specific retention of 3H-E2 in cells by ERs in cells was assessed by the incubation of cells with 10−6 M ICI. The graph represents the mean ± SEM of three independent experiments performed in duplicate. e MDA-MB-231 cells were infected with recombinant adenoviruses in the absence of E2 for 48 h. Cells were then incubated in fresh medium with (E2) or without (−) 10−9 M E2 for 24h. Total RNA was subjected to qPCR. Results for the expression of the CCNA1 (Cyclin A1), TGFA (Transforming growth factor α), CRISPLD2 (Cysteine-rich secretory protein LCCL domain-containing 2), C3 (Complement component 3), TFF1 (Trefoil Factor 1), CTGF (Connective tissue growth factor), PLAT (tissue-type plasminogen activator), IL1B (Interleukin 1b), DKK1 (Dickkopf-1), and HAS2 (Hyaluronan synthase-2) genes are the mean ± SEM of three independent determinations in duplicate. Responses are given as fold change compared to response observed with cells infected with the parent Ad5 (600 MOI) in the absence of E2, which was set to one. Superscript (a) denotes responses that are significantly different from responses observed with cells infected with Ad5 in the absence of E2. Superscript (b) indicates responses that are significantly different from those observed with cells synthesizing ERs in the absence of E2. Superscript (c) indicates responses that are significantly different from responses to ERβ + E2