The LS174T, LOVO, HT29 and HCT116 CRC cell lines were obtained from ATCC (Virginia, USA). SW1222 CRC cells were a kind gift by Prof. W. Bodmer, Department of Medical Oncology, Weatherall Institute, Oxford, UK. HT29 cells with low and high metastatic potential (HT29lm and HT29hm, respectively) were a kind gift of Dr. Raffaella Giavazzi, Institute Mario Negri, Milano, Italy. CCD-18Co human normal colon fibroblasts were from ATCC. All cell lines were cultured in Dulbecco's Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Gibco, Invitrogen, Life Technologies, Carlsbad, CA, USA) at 37 °C in 5% CO2 atmosphere. The TGFBI-silenced SW1222 cell line was generated using anti-TGFBI shRNA-expressing lentiviral particles. Anti-TGFBI shRNAs were from Sigma Aldrich (St. Louis, MO, USA; cat. no. TRCN0000062177 (#1) and TRCN0000062175 (#2)). Control shRNA (shNT) was an anti-eGFP shRNA plasmid (Sigma; cat. no. SHC005). All shRNAs were inserted in the pLenti6/V5 vector using the pLenti6/V5 Directional TOPO® Cloning Kit (Invitrogen, Carlsbad, CA, USA, Part # K4955-00). ShRNA-expressing lentiviral vectors were co-transfected in Lenti-X™ 293T cells (Clontech, Mountain View, CA, USA; Part # 632180) with the pLenti6-Luciferase, psPAX2 (Addgene, Cambridge, MA, USA; Part #12260) and pVSV-G plasmids. Viral supernatants were collected at 48 h - 96 h post-transfection, and filtered (0.45 µm). SW1222 cells were incubated with these lentiviral particles for 48h and then selected by incubation with 1 µg/mL puromycin (Sigma Aldrich, St. Louis, MO, USA). Primary human umbilical vein endothelial cells (HUVECs) were used at early passages (passages II-V), and grown on plastic surface coated with porcine gelatin in M199 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 20% fetal calf serum (FCS) (Invitrogen), 100 µg/mL endothelial cell growth factors (Sigma-Aldrich, St. Louis, MO, USA), and 100 µg/mL porcine heparin (Sigma-Aldrich, St. Louis, MO, USA). CTCs and primary cancer cells (CPP) from primary and metastatic CRC biopsies were isolated, and established as previously described 19, 20. They were maintained in ultralow attachment 24-well plates (Corning) with 1 mL of M12 medium that included DMEM-F12 (Gibco), 2 mmol/L of L-glutamine (Gibco, Thermo Fisher Sci., Waltham, MA, USA), 100 unit/mL of penicillin and streptomycin (Gibco, Thermo Fisher Sci., Waltham, MA, USA), N2 supplement (Gibco, Thermo Fisher Sci., Waltham, MA, USA), 20 ng/mL of epidermal growth factor (R&D Systems, Minneapolis, MN, USA) and 10 ng/mL of fibroblast growth factor-basic (R&D Systems, Minneapolis, MN, USA).

Conditioned medium (CM) from CRC cell lines was obtained after 48h incubation of 80% confluent cells in serum-free medium. CM were collected, centrifuged at 150×g, RT, for 5 min, and then added to CCD-18Co cell monolayers (cells were pre-starved in serum-free medium for 6h) for 48h. Then, fibroblast monolayers were washed with PBS twice and lysed for western blot analysis. For incubation with recombinant human TGF-β1 (Roche, catalog no. 11412272001), 80% confluent cells were starved in serum-free medium for 16h and then incubated with 5 ng/ml of recombinant TGF-β1 in serum-free medium for 48 h. Medium with TGF-β1 was refreshed after 24 h.

Human anti-SMAD2 siRNA (ON-TARGETplus SMARTpool Human SMAD2 (4087)) and scramble siRNA (ON-TARGETplus NonTargeting Control Pool, catalog no. D-001810-10-05) were from Dharmacon. SW1222 cells were transfected with 20 nM of each siRNA using Lipofectamine (Lipofectamine 2000 reagent, catalog no. 11668-019, Life Technologies, Carlsbad, CA, USA).

When indicated, the following compounds were used: SB202190 (5 µM, catalog no. S7067, Sigma-Aldrich), BAY11-7082 (5 µM, catalog no. B5556, Sigma-Aldrich), SP600125 (5 µM, catalog no. S5567, Sigma-Aldrich), MK2206 (1 µM, catalog no. 1032350-13-2, Santa Cruz Biotechnology, Dallas, TX, USA), PD98059 (5 µM, catalog no. 19-143, Merck Millipore, Burlington, MA, USA), ARRY-614 (10 µM, catalog no. S7799, Selleckchem), and LY2228820 (5 µM, catalog no. A413122, Sigma-Aldrich).

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