Transwell 12 well plates

Transwell 12 well plates (8μm pores, Corning, NY, USA) were coated with fibronectin (Sigma) to facilitate migration were seeded with 5 × 10⁴ colon cancer cells per well. Following an incubation period of 6h, non-migrated cells at the membrane top were removed and the cells that have migrated through the membrane were fixed with 4% PFA followed by DAPI (nuclear stain) staining. Images were captured by fluorescent microscopy and the number of migrated cells per well was determined by counting the cells at the center of each well using image J software to digitized images, reduce background and quantify number of particles detected. Data are expressed as number of particles counted per high powered field (hpf).

Migration assays were performed as previously described^{13 (link), 32 (link)}. Briefly, transwell 12 well plates (8 µm pores, Corning) with fibronectin (Sigma, St. Louis, MO) were seeded with 5 × 10⁵ colon cancer cells per well. Cells were treated with either activin A (10 ng/ml) or TGF-β (10 ng/ml) or follistatin (100 ng/ml) for 30 minutes and TGF-β (10 ng/ml) was added. Cells were then allowed to migrate for 6 hours, stained, and imaged. Images from 5 microscopic fields at the center of each well were counted using ImageJ software^{48 (link)}.

Icaritin (purity ≥ 98.5%) and remdesivir (purity ≥ 98.5%) were provided by Beijing Shenogen Pharmaceutical Co. Ltd. (Beijing, China). Diosmin (97% purity), ibuprofen (purity ≥ 99%), dimethylsulfoxide-d⁶ (d⁶-DMSO), D₂O, Cy5 NHS and Cy5.5 NHS were purchased from Beijing Inno-chem Co. Ltd. (Beijing, China). Poloxamer 407 (P407) and Soluplus^® were obtained from Beijing Fengli Jingqiu Pharmaceutical Co. Ltd. (Beijing, China). Chlorpromazine hydrochloride (CMZ), amiloride, genistein, methyl-β-cyclodextrin (MβCD) and β-glucurondase were gained from Sigma-Aldrich (St. Louis, MO, USA). Hank’s balanced salt solution (HBSS), Dulbecco’s modified Eagle’s medium (DMEM), penicillin–streptomycin, trypsin-EDTA were all purchased from Beijing M&C Gene Technology (Beijing, China). Fetal bovine serum (FBS) was supplied by GIBCO (Burlington, Ontario, Canada). Plastic culture flasks (25 and 75 cm²) and Transwell 12-well plates were purchased from Corning (Corning, NY, USA). All other chemicals used were of the highest purity available.