Fibroblast growth factor 2 (fgf2)

Growth Factor rescue experiments were performed in DiFi and LIM1215 colorectal cancer cell lines treated with CET (provided by Merck KG), AMG-337 and BGJ-398 (Selleckchem), FGF1, FGF2, TGFβ1, TGFβ2 and TGFβ3 (RnD Systems) and HGF and FGF10 (Peprotech) for 5 days (7 days for FGF10). Treatments were replenished with fresh media after 3 days in 7 day assays. EGFR mutant experiments were performed in LIM1215 cells. Cells were treated with CET for 5 days. DiFi and LIM1215 cells were seeded in standard media or CAF CM and treated with CET for 5 days. All experiments were performed in 6 replicates. Viability was assessed using CellTiter Blue reagent (Promega) for all assays.

Mouse primary fibroblasts were isolated6 (link) and cultured in DMEM supplemented with 10% FCS or Amniomax (Gibco). Cells were pulsed with 10 μM EdU 3–4 h before fixation and staining with the Click-it EdU Imaging Kit (Invitrogen). To stimulate or inhibit specific signalling pathways in fibroblasts, the following concentrations of GFs and inhibitors were added to the medium for 24 or 48 h: Shh 1 μg ml⁻¹; TGF-β2 10 ng ml⁻¹; FGF2 20 ng ml⁻¹; FGF5 10 ng ml⁻¹ (all from RnD Systems; vehicle: PBS); IPI4182 0.5 μM (Infinity Pharmaceuticals; vehicle: DMSO); RepSox 25 μM (Tocris; vehicle: DMSO); PD173074 2 μM (Tocris; vehicle: DMSO). DED was prepared by floating 5 mm diameter punch biopsies of back skin on 0.8% trypsin (Gibco) dissolved in PBS for 60 min, separating the dermis from the epidermis with forceps and depleting cells from the tissue by at least ten freeze–thaw cycles. Before fibroblasts were seeded onto DEDs, the tissue was placed into 24-well cell culture inserts and equilibrated with medium.