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Figure 8 | Breast Cancer Research

Figure 8

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

Figure 8

Transforming growth factor β downregulates BCAR3 in a Smad-dependent manner. (a) Basal-like breast cancer cells were starved and treated with 200 pM transforming growth factor β (TGFβ) for 24 hours. Levels of BCAR3 protein expression were examined by Western blot analysis. (b) MDA-MB-231 cells were transfected with Smad2 small interfering RNA (siRNA) or Smad3 siRNA for 48 hours, then starved and treated with 200 pM TGFβ for 24 hours. Levels of BCAR3 protein expression were examined by Western blot analysis. (c) The signal intensity of BCAR3 was quantified and normalized to that of β-tubulin. The results show normalized signal intensity calculated from three independent experiments, and error bars show standard error of the mean. An asterisk indicates statistical significance (P < 0.05) as determined by unpaired Student’s t-test. (d) and (e) MDA-MB-231 (d) or BT-549 (e) cells were treated with or without 100 pM TGFβ for 24 hours. Total RNA was extracted, reverse-transcribed and subjected to real-time PCR to examine expression of connective tissue growth factor (CTGF) and BCAR3. The results show average fold changes calculated from three independent experiments, and error bars show standard error of the mean. An asterisk indicates statistical significance (*P < 0.05) as determined by unpaired Student’s t-test. (f) MDA-MB-231 cells were starved and treated with 10 μM MG-132 or DMSO as vehicle control for 1 hour, then stimulated with or without 200 pM TGFβ for 24 hours, as indicated. BCAR3 protein levels were examined by Western blot analysis (left panel). Relative BCAR3 protein levels, compared to β-tubulin, were quantified by densitometry for two separate experiments. DMSO, Dimethyl sulfoxide. (g) Model of BCAR3 in mediating a positive feedback loop downstream of the TGFβ/Smad signaling axis.

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