Ccd 18co

Primary (SW480, ATCC^® CCL-228 ™) and metastatic (SW620, ATCC^® CCL-227 ™) colon cancer cell lines and normal colon cell line (CCD-18co, ATCC^® CRL-1459 ™) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). SW480 and SW620 cells were maintained in Leibovitz L-15 medium (Corning Life Sciences, Corning, NY, USA) supplemented with 10% fetal bovine serum and 1% antibiotics; penicillin and streptomycin, in a 37 °C incubator without CO₂. CCD-18co cells were maintained in Eagles Minimum Essential Medium (EMEM) (Corning Life Sciences, USA) supplemented with 10% fetal bovine serum and 1% antibiotics, in a 37 °C incubator with CO₂. Cells were frequently monitored to ensure a normal and consistent morphology. While maintaining proper aseptic techniques, the cells were subcultured every 2–3 days or upon 90% confluency and the culture medium replenished within a biosafety cabinet (Labculture © Class II, Type A2 Biological Safety, Esco Technologies Inc., Horsham, PA, USA).

The primary normal fibroblast PHBF (Bladder), CCD-18Co (Colon) and HOF (Ovary) were serum-starved overnight before treatment with media only (untreated), 10 ng/mL rhTGFβ1 or 10 ng/mL rhTGFβ1 + 10 µM Galunisertib for 24 h, and the total RNA was extracted for RNA-seq analysis as previously described^{8 (link)}. To detect IL-6 protein in the supernatant, cells were treated for 48 h with recombinant human TGFβ1. After the 48-h timepoint, the supernatant was collected and analysed by Luminex using the Millipore kit. For the proliferation assay, PHBF, CCD-18Co, HOF were plated at 3000 cells/well in a 96-well culture flat bottom plate for immunofluorescence assays (Corning, #3917) overnight. Cells were then cultured for 72 h in DMEM high glucose + 1% FBS with the indicated concentration of TGFβ1 with or without Galunisertib. Next, CellTiter-Glo^® reagents (Promega, #G7570) were added to each well, and the luminescence signal was read with a microplate reader.