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Figure 3 | Breast Cancer Research

Figure 3

From: Breast cancer anti-estrogen resistance 3 inhibits transforming growth factor β/Smad signaling and associates with favorable breast cancer disease outcomes

Figure 3

BCAR3 antagonizes function of canonical transforming growth factor β signaling. (a) SCP2 cells were cotransfected with a (CAGA)12-lux luciferase reporter construct, constitutive β-galactosidase construct and 50 pM scrambled small interfering RNA (siRNA) control or a pool of siRNAs targeting BCAR3 (25 pM each). Cells were starved overnight, stimulated with or without 100 pM transforming growth factor β (TGFβ) and subjected to luciferase assays. Luciferase activities were normalized by β-galactosidase activity and are represented as relative luciferase units (RLUs). Error bars show standard errors of the mean of three independent experiments. An asterisk indicates a statistically significant difference as determined by unpaired Student’s t-test (*P < 0.05). The inset under the figure shows basal luciferase activities. (b) SUM-159-PT cells were cotransfected with (CAGA)12-lux luciferase reporter construct, constitutive β-galactosidase construct and FLAG-tagged AND-34. Luciferase assays were performed as described in (a). The inset under the figure shows basal luciferase activities. (c) Inducible MCF-7 cells were cultured with or without doxycycline (Dox) for 72 hours, starved overnight and stimulated with 100 pM TGFβ for 24 hours. mRNA levels of three Smad-dependent genes (connective tissue growth factor (CTGF), transmembrane prostate androgen induced RNA (TMEPAI) and Smad7) were examined by real-time PCR. Bars show fold changes of induction by TGFβ. Error bars show SEM of three independent experiments. An asterisk indicates a statistically significant difference as determined by unpaired Student’s t-test (*P < 0.05). (d) Inducible MCF-7 cells were cultured with or without Dox for 72 hours, starved overnight and stimulated with 100 pM TGFβ for 72 hours. Cell viability was examined by using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; thiazolyl blue tetrazolium bromide) assay. Error bars show SEM of three independent experiments. An asterisk indicates statistically significant difference (*P < 0.05).

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