Tgfβ1

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.

For the treatment with 0–50 ng/mL BDNF [13 (link)] or with 1 ng/mL TGF-β1 [20 (link)] (RnD Systems, Minneapolis, MN, USA), 6.7 × 10⁴ cells/mL were plated in serum containing medium [20 (link)] and cultured for 72 h. After that, the cells were washed with PBS and incubated with 10 µg/mL Mitomycin C (Sigma–Aldrich^®, St. Louis, MI, USA) in serum-free albumin-containing medium for 30 min at 37 °C ensuring cell cycle arrest [40 (link)]. Then the cells were washed twice with albumin-containing medium and subsequently treated with albumin-containing medium for two times 48 h supplied with 25 ng/mL recombinant human BDNF [13 (link)] for altogether 96 h. After completion of treatments, the cells were used for cell counting with trypan blue staining (Sigma, Darmstadt, Germany) in a Neubauer chamber (Paul Marienfeld GmbH & Co. KG, Lauda-Königshofen, Germany). One-thousand cells from all samples were plated in 75-cm² cell culture flasks grown in serum-supplemented medium for 3 weeks and the growing colonies were stained with gentian violet and were counted. The number of growing colonies were related to plated cell numbers (1000) [57 (link)].
During the treatments cells were filmed by a Juli BR live cell imaging system (Peqlab, Erlangen, Germany), which was also used for estimation of the percentage of covered area in the culture dish.

Human primary renal proximal tubular epithelial cells were purchased from Innoprot (Derio, Spain) and American Type Culture Collection (ATCC, Manassas, USA) and were maintained in Renal Epithelial Cell Basal Medium (ATCC) supplemented with Renal Epithelial Cell Growth Kit Components (ATCC). Human primary renal fibroblasts were purchased from Innoprot and maintained in DMEM F12 (Invitrogen) containing 10% FCS and supplemented with 2 mM L-Glutamine. For experiments, epithelial cell medium was used for both cell types. Cells were grown in 100% humidity and 5% CO₂ at 37°C and were used until Passage 6. For hypoxia experiments, cells were grown in 2.5%O₂ and 5% CO₂ using a Panasonic MCO-19 M incubator. For stimulation with cytokines, cells were incubated with TGFβ1 10 ng/ml (RnD Systems, Minneapolis, USA) added at the start of the culture. Aristolochic acid and H₂O₂ were from Sigma and were used at the indicated concentrations. For TGF-β blockade, cells were incubated with anti-TGF-β MAB1835 or appropriate isotype control (RnD Systems) for the duration of the culture period.