Pd166866

Stock solutions of 20 mM PD166866 (Sigma-Aldrich, PZ0114) and 10 mM DMH1 (Sigma-Aldrich, D8946) were prepared in DMSO and aliquots were stored at −20°C until use. Treatment solutions were freshly prepared by diluting stock solutions in embryo medium and kept at 28.5°C. The final test concentrations of 8 µM PD166866 and 12 µM DMH1 were selected based on previous studies (Haerlingen et al., 2019 (link)). Embryos treated with 0.1% DMSO served as a vehicle control group for all small molecule treatment experiments. Media were supplemented with 0.01 mg/ml Methylene Blue to prevent bacterial growth and 0.003% PTU to prevent pigmentation.
Two hours before treatment initiation, embryos were staged and stage-matched embryos were pooled and randomly allocated to the different experimental treatments into 60 mm Petri dishes (40-50 maximum per dish). Treatment periods are indicated in the text and in the corresponding figures. All treatments were performed in duplicate. All incubations of embryos in small molecule solutions were performed at 28.5°C in the dark. After completion of small molecule treatment, embryos were washed three times with fresh embryo medium before transfer into clean 96 mm dishes filled with fresh embryo medium.

22Rv1 and VCaP cell lines were obtained from the American Type Culture Collection (Manassas, VA) and authenticated at this site. Cells were maintained in RPMI-1640 medium supplemented with 10% FBS. 22Rv1 stable transfectants were generated using the CR-1 cDNA cloned into the p3XFLAG-Myc-CMV-25 expression vector (Sigma Aldrich, St Louis, MO), or empty vector, and transfection by Lipofectamine 2000 (Life Technologies, Grand Island, NY) followed by G418 selection at 400 μg/mL for three to four weeks. LY294002 and U0126 were purchased from LC Laboratories (Woburn, MA), PD166866 and SB431542 were from Sigma Aldrich.

Human atrial fibroblasts were purchased from Lonza Research Laboratory (Walkersville, MD, USA). As described previously [22 (link)], cells were seeded on uncoated culture discs as monolayers and cultured in Dulbecco’s modified Eagle medium (Thermo Fisher Scientific, Loughborough, UK) containing 10% fetal bovine serum (Hyclone, Logan, UT, USA) and 100 U/mL penicillin–streptomycin (Thermo Fisher Scientific, Loughborough, Gibco, UK) in a humidified atmosphere of 5% CO₂ at 37 °C. Cells used in this study were from passages 4–6 to avoid possible variations in cellular function. Cardiac fibroblasts treated without and with recombinant mouse FGF-23 (1, 5 or 25 ng/mL; R&D Systems, Abingdon, UK) in the presence and absence of an FGF receptor 1 antagonist (PD166866, 1 μM; Sigma, St. Louis, MO, USA), a free Ca²⁺ chelator (EGTA, 1 mM; Sigma), or a PLC inhibitor (U73122, 1 μM; Abcam, Cambridge, UK) for 48 h were harvested for further analysis.

Cells were cultivated under standard conditions (37 °C, 5% CO₂) and grown in melanoma isolation media (MIM): MCDB153 medium / Leibovitz’s L-15 medium (4/1) supplemented with 2% FBS, 7.5% NaHCO₃, 5 µg/ml Insulin, 5 ng/ml EGF, 1.68 mM CaCl₂, 50 mg/L streptomycin sulphate and 30 mg/L penicillin. Cells were split when 90% confluent using a 0.25% trypsin/EDTA solution. If not otherwise indicated, experiments were performed in MIM for 48 hours. 4-phenylbutyric acid (4-PBA) was obtained from Santa Cruz Biotechnology (Dallas, USA) and PD166866 and thapsigargin were obtained from Sigma-Aldrich (St. Louis, USA).

Cells were pre-treated with 4-PBA and PD166866 (Sigma-Aldrich) and maintained in MIM for 16 hours. Afterwards cells were detached and re-suspended in MIM without FBS and 0.1% BSA containing the described treatments. 25.000 cells were then seeded onto matrigel invasion chambers (Corning, New York, USA), which were prepared according to the manufacturers protocol. After 8 hours, cells were fixed with formaldehyde (Sigma-Aldrich), stained with crystal violet (Sigma-Aldrich) and invading cells were counted under the microscope.

Defined compounds used for validation experiments were ordered de novo from various suppliers: SB-431542, PD-173074, U0126, Salinomycin, Nigericin, PD-161570, PD-166285, PD-166866, Idoxuridine and 5-Azacytidine (all from Sigma); Y-27632 2HCl, GSK429286A, Nintedanib, Pazopanib, Sorafenib, Vatalanib, Axitinib, Sunitinib, Erlotinib, Lapatinib, Gefitinib, Dasatinib, Crizotinib, Vandetanib, AEE788, Cediranib, AZD4547, CP673451, TSU-68 and KX2-391 (all from Selleck Chemicals); PP1, PP2, Rho kinase inhibitor V, SR-3677, GSK269962, SB-772077B (all from Tocris); Fasudil from Enamine; CAY10622 and PD-166326 from Cayman; BGJ398 from Axon Medchem; 5-Aza-2′-deoxycytidine from Chem-Impex. The reordered compounds were tested in extended dilution series ranging from 25 μM to 0.04 nM on TGFβ-treated NMuMG cells and from 25 μM to 1 nM on TGFβ-treated Py2T cells.