Smad2 3

Whole cell lysates were prepared in RIPA buffer (Sigma) and quantified using Bradford Protein Assay (Bio-Rad). Twenty micrograms of lysates were resolved on Bolt^® 4–12% Bis-Tris Plus gels using Bolt^TM MOPS SDS Running buffer and transferred to a Hybond C super nitrocellulose membrane (GE Healthcare). After blocking to prevent non-specific binding in 5% milk in PBST for 1 h at room temperature, membranes were incubated with the specific primary antibodies overnight at 4 °C. The following primary antibodies were used: E-cadherin (Takara Bio Inc., M106, clone HECD-1, 1:1000 dilution), Occludin (Cell Signaling, #5446, 1:1000 dilution), Vimentin (Cell Signaling, #5741, 1:3000 dilution), ZEB1 (Santa Cruz, sc-25388, 1:500 dilution), SNAI1 (Santa Cruz, sc-28199, 1:500 dilution), LIN28B (Cell Signaling, #4196, 1:1000 dilution), LIN28A (Cell Signaling, #3978, 1:1000 dilution), SMAD2/3 (Cell Signaling, #8685, 1:1000 dilution), β-actin (Abcam, ab8227, 1:200,000 dilution), GAPDH (Santa Cruz, sc-137179, 1:10,000 dilution). Following incubation with the specific HRP-conjugated antibody (Dako, #P0447 or #P0448, 1:2,500 dilution), chemiluminescence signal was detected using Amersham^TM ECL^TM Western blotting detection reagents (GE Healthcare) and Amersham Hyperfilm^TM ECL (GE Healthcare). Uncropped scans of the most important blots are shown in Supplementary Fig. 13.

Metformin and phenformin were purchased from Sigma-Aldrich (St. Louis, MO, USA); human TGF-β1 and antibodies against β-actin, p-AMPKα Thr172, AMPKα, p-ACC Ser79, ACC, Slug, p-Smad3 Ser423/425, and Smad2/3 were from Cell Signaling Technology (Beverly, MA, USA); ELISA kits for human and mouse TGF-β1, antibodies against E-cadherin, and vimentin were from Abcam (Cambridge, MA, USA); monoclonal antibody against LKB1 was from Santa Cruz Biotechnology (Santa Cruz, CA, USA); growth factor-reduced Matrigel and monoclonal antibody against β-catenin were from BD Biosciences (San Jose, CA, USA); Lipofectamine 2000, 4′,6-diamidino-2-phenylindole (DAPI), fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit antibodies were from Life Technologies (Grand Island, NE, USA); Chamber slides was from EMD Millipore (Billerica, MA, USA).

The following antibodies were used in this study: DCP1A (Santa Cruz, 56-Y), SYK (Santa Cruz, N19), G3BP1 (BD Biosciences, 611126), LC3-A/B (Cell Signaling Technology, D3U4C), p62 (Abcam, ab56416), E-cadherin for immunoblot (Santa Cruz, H10), DDX6 (Sigma Aldrich, P0067), GAPDH (Ambion, AM4300), SMAD2/3 (Cell Signaling Technology, 3102), phospho-SMAD2 (Cell Signaling Technology 3101), E-cadherin for IHC (BD biosciences, 610182), Ki67 (BD biosciences, 550609), AlexaFluor 594-conjugated goat anti-rabbit IgG (Invitrogen), AlexaFluor 594-conjugated goat anti-mouse IgG (Invitrogen), and AlexaFluor 488-conjugated goat anti-mouse IgG (Invitrogen), biotin-conjugated goat anti-mouse IgG (Jackson). For immunohistochemistry biotinylated secondary antibodies were detected using the ABC elite kit in combination with 3-3′-diaminobenzidine (Vector).

Protein extraction and Western blotting experiments were performed as described previously [12 (link)]. Primary antibodies used were as follow: anti-TGF-β1, CDK4, c-Myc, CyclinD3, p27, phospho-Akt, Akt, phospho-Erk1/2, Erk1/2, phospho-JNK, JNK, phospho-p38, p38, phospho-Smad 2, Smad 2/3, Smad 4, and Histone H3 (Cell Signaling, Danvers, MA); anti-CD63, CD81, and CD9 (Santa Cruz, Santa Cruz, CA); and anti-β-actin (Sigma-Aldrich). HRP-conjugated horse anti-mouse and goat anti-rabbit IgG (Cell Signaling) were used as secondary antibodies.