β-Catenin knockdown chimera F-TrCP-Ecad abolishes TGF-β1-induced E-cadherin promoter repression, Snail transcription and MMP-9 expression in C1.1 cells. (A) Relative E-cadherin promoter (−201 to +131) activity in C1.1 cells of control, pcDNA3 or F-TrCP-Ecad transfection treated with TGF-β1 (3 ng/ml) for 24 hours, and F-TrCP-Ecad transfection alone. Values are means±s.d. **P<0.01 vs TGF-β1 with or without vector control (pcDNA3), n = 6. (B) The expression of Snail in C1.1 cells with corresponding treatments was analyzed by real-time PCR. Values are means±s.d. **P<0.01 vs TGF-β1 with or without vector control (pcDNA3), n = 6. (C) Representative MMP-9 gelatin zymography of supernatants from C1.1 cells with corresponding treatments. (D) Quantitative analysis of (C). Relative MMP-9 zymograph intensity was calculated against untreated medium. Values are means±s.d. **P<0.01 vs TGF-β1 with or without vector control (pcDNA3), n = 6.

F-TrCP-Ecad promotes soluble nuclear/cytosolic β-catenin degradation. (A) Western blot analysis for E-cadherin and F-TrCP-Ecad (Flag) in C1.1 cells untreated or treated with TGF-β1 (3 ng/ml) for 24 hours, or transfected with pcDNA3 or F-TrCP-Ecad. Values are means±s.d. in relative densitometry intensity. *P<0.05 vs untreated group, **P<0.01 vs E-cadherin, n = 3. (B) Western blot analysis for total β-catenin in C1.1 cells untreated, treated with TGF-β1 (3 ng/ml) for 24 hours, or transfected with pcDNA3 or F-TrCP-Ecad. Values are means±s.d., n = 3. (C) Western blot analysis of β-catenin levels in membrane protein fraction extracted from C1.1 cells untreated, treated with TGF-β1 (3 ng/ml) for 24 hours, or transfected with vector control (pcDNA3) or F-TrCP-Ecad alone. α1 Na/K-ATPase (ATPase) was used as a membrane protein control. Values are means±s.d. *P<0.05 vs the other three groups, n = 6. (D) Western blot analysis of β-catenin levels in cytoplasmic fraction extracted from C1.1 cells untreated, or treated with TGF-β1 (3 ng/ml) for 24 hours and transfected with vector control (pcDNA3) or F-TrCP-Ecad, or transfected with F-TrCP-Ecad alone. Heat shock protein 90 (HSP) was used as the cytoplasmic protein control. Values are means±s.d. *P<0.01 vs control, ^#P<0.05 vs TGF-β1 with or without vector control (pcDNA3), n = 6.

Knockdown of cytosolic β-catenin inhibits TGF-β1-induced EMT through β-catenin/P-Smad3 but not β-catenin/LEF-1. (A) Co-immunoprecipitation (IP) of β-catenin, P-Smad3, P-Smad2 and LEF-1 from C1.1 cell nuclear proteins with rabbit anti-P-Smad3 antibody. Purified rabbit IgG was used as a negative control. Immunoprecipitates were analyzed by western blotting with anti-β-catenin, anti-P-Smad3, anti-P-Smad2 and anti-LEF-1 antibodies. Input refers to analysis of nuclear proteins before immunoprecipitation. Quantification of immunoblots (relative intensity) is shown by fold increase in densitometry intensity (the intensity of the control is arbitrarily defined as 1). Values are means±s.d. *P<0.05 vs TGF-β1 treatment group, n = 3. (B) Co-immunoprecipitation of β-catenin, LEF-1, P-Smad3 and P-Smad2 from C1.1 cell nuclear proteins with rabbit anti-LEF-1 antibody. Purified rabbit IgG was used as a negative control. Immunoprecipitates were analyzed by western blot with anti-β-catenin, anti-LEF-1, anti-P-Smad3 and anti-P-Smad2 antibodies. Values are means±s.d. *P<0.05 vs TGF-β1 treatment group, n = 3. (C) Interactions between β-catenin and LEF-1 were shown by TOP-flash assay. The relative luciferase activity in C1.1 cells transfected with FOP-flash and TOP-flash vectors are shown in control (Ctr), TGF-β1-treated, TGF-β1-treated and F-TrCP-Ecad-transfected, TGF-β1- and ICG-001-treated, LiCl-treated positive control, and ICG001-alone-treated or F-Trcp-Ecad-alone-transfected C1.1 cells. Values are means±s.d. **P<0.01 vs the other six groups, n = 6. There was no statistical difference in TOP-flash activity among the Ctr, TGF-β1-only and TGF-β1 with F-TrCP-Ecad or ICG-001 groups (P>0.05). (D) SMAD reporter assay in C1.1 cells. The SMAD reporter relative response luciferase activities in C1.1 cells are shown in control (Ctr), TGF-β1-treated, TGF-β1-treated and F-TrCP-Ecad-transfected or ICG-001-treated, and ICG001-alone-treated or F-Trcp-Ecad-alone-transfected C1.1 cells. Values are means±s.d. *P<0.05 vs TGF-β1-only group, n = 6.

β-Catenin is dispensable for macrophage activation, and F-TrCP-Ecad or ICG-001 has no effect on anti-inflammatory effects of TGF-β1 in macrophages. (A) Total β-catenin protein levels in J774 cells stimulated by LPS (500 ng/ml) in the absence or presence of TGF-β1 for 24 hours, or treated with TGF-β1 alone. Quantification is shown by relative densitometry intensity. Values are means±s.d. (B) Total β-catenin protein levels in J774 cells stimulated by IFN-γ (20 ng/ml) in the absence or presence of TGF-β1 (10 ng/ml) for 24 hours, or treated with TGF-β1 alone. (C) and (D) Western blot analysis for total β-catenin levels and F-TrCP-Ecad expression (Flag) in J774 cells untreated, treated with TGF-β1 (3 ng/ml) for 24 hours with or without transfection of pcDNA3 or F-TrCP-Ecad, or transfected with F-TrCP-Ecad alone. Values are means±s.d. *P<0.05 vs other groups, n = 6. (E) Real-time PCR analysis of TNF-α expression in J774 cells stimulated by LPS in the presence of TGF-β1 with F-TrCP-Ecad or WT β-cat transfection or ICG-001 treatment, or treated with TGF-β1 or ICG-001 alone, or transfected with F-TrCP-Ecad alone. Values are means±s.d. **P<0.01 vs the other eight groups, n = 6. (F) Real-time PCR analysis of iNOS expression in J774 cells stimulated by IFN-γ in the presence of TGF-β1 with F-TrCP-Ecad or WT β-cat transfection or ICG-001 treatment, or treated with TGF-β1 or ICG-001 alone, or transfected with F-TrCP-Ecad alone. Values are means±s.d. **P<0.01 vs the other eight groups, n = 6.