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Tian et al. EMT drives anti-estrogen resistance in breast cancer

Figure 2: Induction of EMT programs by TGF-β promotes ER-α accumulation in the cytoplasm of MCF-7 cells. (A) MCF-7 cells were
treated with either TGF-β1 (5 ng/mL), estradiol (1 nmol/L), or both agonists for 96 h as indicated. Afterward, the cells were fixed in
paraformaldehyde and processed for indirect E-cad immunofluorescence. Images are representative of 3-independent experiments (×400).
(B and C) MCF-7 cells were treated with TGF-β1 (5 ng/mL) in either 2D- (B) or 3D- (C) cultures for 0-96 h, at which point differences
in ER-α expression were measured by real-time PCR. Data are the mean fold-changes (± SE; n = 3; *P < 0.05; Student’s t-test). (D)
Differential mRNA expression of ER-α and ER-β in quiescent MCF-7 cells. Data are the mean (± SE; n = 3; *P < 0.05; Student’s t-test).
(E) MCF-7 (top) and BT474 (bottom) cells were stimulated with TGF-β1 (5 ng/mL) for 0-96 h. Afterward, ER-α expression levels were
monitored by immunoblotting. Images are representative of 4-independent experiments. (F) MCF-7 cells were transiently transfected
overnight with either the pSBE- or pERE-luciferase reporter genes, as well as with the pCMV-β-galactosidase reporter gene to control for
differences in transfection efficiency. Afterward, the transfectants were stimulated overnight either singly or in combination with TGF-β1
(T; 5 ng/mL) or estradiol (E; 0.1 nmol/L) prior to measuring luciferase and β-gal activities. Data are the mean fold-changes (± SE; n = 3;
*P < 0.05; Student’s t-test). (G) Pre- and post-EMT MCF-7 cells were transiently transfected overnight with pERE-luciferase and pCMV-β-
gal-luciferase cDNAs, followed by 24 h treatment with either TGF-β1 (5 ng/mL) or estradiol (0.1 nmol/L). Data are the mean fold-changes
(± SE; n = 3; *P < 0.05; Student’s t-test). (H) MCF-7 cells were treated with TGF-β1 (5 ng/mL) for 0-96 h, at which point the expression of
ER-α in cytoplasmic and nuclear cell fractions was determined by immunoblotting. Stripped blots were reprobed with antibodies to either
HDAC1 or β-actin to monitor integrity of nuclear and cytoplasmic fractions, respectively. Images are representative of at least 3-independent
experiments. TGF: transforming growth factor; EMT: epithelial-mesenchymal transition; ER: estrogen receptor; SE: standard error
Journal of Cancer Metastasis and Treatment ¦ Volume 3 ¦ August 21, 2017 155