TGFβ1 signaling in primary fibroblasts

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.