Figure 4: Mutation of TGFβ receptors results in loss of function.
From: Inactivation of TGFβ receptors in stem cells drives cutaneous squamous cell carcinoma
(a) Indicated TGFBR1 plasmids were co-transfected into TGFBR1-null mouse embryonic fibroblasts and assayed for SMAD7-Promoter Luciferase (SMAD7 Promoter-Luc) reporter gene activity and receptor expression levels by western blotting (lower panels) with and without TGFβ stimulation for 4 h. β-Actin is used as a loading control. EV, empty vector control; WT, wild type. Data are mean ±s.d., n=3. (b) Indicated TGFBR2 plasmids were co-transfected into TGFBR2-null T47D cells and assayed for SMAD7-Promoter Luciferase (SMAD7 Promoter-Luc) reporter gene activity and receptor expression levels by western blotting (lower panels) with and without TGFβ stimulation for 4 h. β-Actin is used as a loading control. EV, empty vector control; WT, wild type. Data are mean ±s.d., n=3. (c) PO4-SMAD3 activity was assessed by IHC in wild-type and mutant tumours (n=8, ***P=0.001, Mann–Whitney U-test). Representative images are shown. Scale bar, 100 μM. (d) Effects of TGFβ stimulation on growth of NHK and cSCC cell lines. Data represent Cell Titre Glo measurement of cell proliferation over the indicated time course of cells treated with the indicated dose of TGFβ1. NHKs and cell lines harbouring mutant TGFBR2 (SCCIC8 and SCCIC12) are shown. Data represent the mean ±s.d. n=6. (e) Restoration of wild-type TGFBR2 restores growth inhibition. SCC1C8 and SCCIC12 cells were co-transfected with empty vector control (EV) or wild-type TGFBR2 expression plasmids (TGFBR2) and a GFP expression plasmid. Proliferation of GFP+ve cells was assessed using real-time Incucyte Zoom imaging over 6 days. Data represent the mean ±s.d. n=6. *P<0.05, **P<0.01 and ***P<0.001 (Student’s t-test).
