Tgfbr1

Sections of formalin-fixed, paraffin-embedded tumors were used. Briefly, after antigen retrieval, the following primary antibodies were used: ITGAV (sc-376,156, Santa Cruz,); ITGB1 (AB3167, Abcam), ITGB6 (HPA023626, Atlas), CEACAM7 (LS-B13068, LSBio), TGFBR1 (PA5–98192, Thermo Scientific), phospho-SMAD2 (AB3849, Millipore), TGFBI (MA526731, Thermo Fisher Scientific), Twist (ab50581, Abcam), Ki 67 (M7240, Agilent); human-specific fibronectin (NCL-FIB, Leica Biosystems); fibronectin, cross-reacting with the fibronectin-equivalent protein in mouse (A0245, Dako); STAT1 (HPA000931, Sigma-Aldrich), HLA-DR (M0746, Agilent). Vectastain ABC kit (Vector) was used. Slides were scanned by Axio Scan Z1 (Zeiss). For quantification with Image J (NIH), images of four randomly chosen vision fields (not containing necrotic areas) were analyzed. If artifacts interfered with automated quantification by Image J, the staining was quantified by two experienced investigators: grade 0: no reaction or weak focal reaction; grade 1 intense focal or diffuse weak reaction; grade 2 moderate diffuse reaction; grade 3 for an intense diffuse reaction.

Transient knockdown experiments were conducted with the human siRNA-SMARTpool of IGF1R, EGFR, TGFBR1 and siControl (Thermo Scientific, Wilmington, DE) for oncogenic receptors; anti-miR inhibitor and anti-Ctl (Invitrogen, Carlsbad, CA) for miR-133a. CL1-5 or A549 cells were transfected with indicated siRNAs or anti-miRNA inhibitor by RNAiFect transfection reagent (Qiagen,Valencia, CA) according to manufacturer's protocols. The cells were used for invasion experiments 48 hours post-transfection and cell numbers were counted 72 hours post-transfection.
BEAS-2B cells were seeded in 6-well plates at a concentration of 1×10⁵ for treatment with 50 µM LY294002 (Sigma-Aldrich, St. Louis, MO). The cells were used for invasion and proliferation assays 48 and 72 hours post-treatment, respectively.
Cells were seeded in 60-mm dishes at a concentration of 2×10⁵ for treatment with rIGF-1 (R&D Systems Inc., MN, USA) or human TGF-β-1 (PeproTech, Rocky Hill, NJ). After serum starvation for 8 hours, CL1-5 and A549 cells were treated with 250 ng/ml rIGF-1 for 30 and 2.5 min, respectively. For TGF-β-1 stimulation, A549 cells were treated with 0.2 ng/ml TGF-β-1 for 5 min.