Figure 5.

TβRIII shedding mutants alter BMP mediated signaling, migration, and invasion in mammary epithelial cells. (A) Western blot analysis of pSmad1/5/8 in HMECs transfected with vector control, TβRIII, ∆shed-TβRIII, or SS-TβRIII. Transfected HMECs were serum starved overnight prior to BMP2 treatment. (B) Vector control, TβRIII, ∆shed-TβRIII, or SS-TβRIII transfected HMECs were plated in a fibronectin transwell migration assay in serum free conditions on a fibronectin coated transwell with and without BMP2 treatment for 24 hours. Data was normalized to vector UT and fold change ± SEM is shown. *P ≤ .05; t test. (C) Western blot analysis of pSmad1/5/8 signaling in MDA-MB-231 monoclonal lenti-stable cell lines. Cells were serum starved overnight and treated for 10 minutes with BMP2. (D and E) Western blot analysis of pSmad1/5/8 signaling in MDA-MB-231-EV, TβRIII, ∆shed-TβRIII, or SS-TβRIII MDA-MB-231 monoclonal stable cell lines. Media was changed to 1mL overnight and cells were treated the next day with BMP2 at the indicated dosages and times. (F) MDA-MB-231-EV, TβRIII, ∆shed-TβRIII, or SS-TβRIII monoclonal stable cell cells were plated in a matrigel transwell invasion with serum free CM (24 hour conditioning) from corresponding cells on a matrigel coated transwell with and without BMP2 treatment assay for 24 hours. Data was normalized to EV UT as well as normalized for proliferation and fold change ± SEM is shown. *P ≤ .05; t test. (G) MDA-MB-231-EV, TβRIII, ∆shed-TβRIII, or SS-TβRIII monoclonal stable cells were plated in a fibronectin transwell migration assay with serum free CM from corresponding cells on a matrigel coated transwell with and without BMP2 treatment. Data was normalized to EV UT as well as normalized for proliferation and fold change ± SEM is shown. *P ≤ .05; t test. β-Actin levels are shown as loading controls for westerns. All experiments were independently performed at least 3 times and representative data are shown.