Icg001

The human NSCLC cell line A549 was obtained from the ATCC and grown in Ham’s F12 (Gibco, Grand Island, NY). Another human NSCLC cell line CL1–1 was kindly provided by Dr. Pan-Chyr Yang (Department of Internal Medicine, National Taiwan University Hospital, Taiwan, R.O.C.) and grown in Dulbecco modified Eagle medium (DMEM) (Gibco). HT29 (human colorectal cancer cell line) was obtained from the ATCC and grown in DMEM. Cells were cultured in F12 or DMEM containing 10 units/ml penicillin, 10 μg/ml streptomycin and 10% fetal bovine serum (FBS; Gibco). For enrichment of CSCs in spheroid culture, cancer cells were suspended in tumor sphere medium consisting of serum-free DMEM/F12, N2 supplement (Gibco), human recombinant epidermal growth factor (EGF) (20 ng/ml, PeproTech, Rocky Hill, NJ), and basic fibroblastic growth factor (bFGF) (10 ng/ml, PeproTech). Cell colonies > 50 μm in diameter and > 50% in area showing 3-dimensional structure and blurred cell margins were defined as spheres. Sphere numbers were counted at day 12 of culture. Treatment reagents included FAK inhibitor (10 μM) (Calbiochem, San Diego, CA), SB216763 (20 μM) (Sigma-Aldrich; St. Louis, MO), LY294002 (10 μM) (Abmole Bioscience, Houston, TX), and ICG001 (10 μM) (Abmole Bioscience).

The normal human kidney immortalized proximal tubule epithelial cell line, HK-2 cells, were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin solution at 37°C in a 5% CO₂ incubator. Under the microscope, the cells were adherent. TGF-β1 is a known fibrosis inducer, and ICG-001 is a known Wnt/β-catenin pathway inhibitor; thus, TGF-β1 could be used to induce EMT in HK-2 cells. ICG-001 can be used in rescue experiments to further confirm the role of the Wnt/β-catenin pathway in renal fibrosis. TGF-β1 was obtained from Abcam (ab50036), and ICG-001 was purchased from AbMole (M2008). To investigate the role of SKI in cell EMT, HK-2 cells were treated with TGF-β1 (10 ng/mL) for 24 h at 37°C. HK-2 cells in the exponential growth phase were digested with 0.25% trypsin-EDTA, passaged, and counted, and the HK-2 cells were inoculated into a six-well plate. Next, either SKI or ICG-001 was mixed with TGF-β1. After 24 h of stimulation, the cells were collected for further analysis.