Fgf19
Manufactured by R&D Systems
Sourced in United States, United Kingdom
FGF19 is a member of the fibroblast growth factor (FGF) family. It functions as a signaling protein involved in various biological processes.
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Lab products found in correlation
Dexamethasone (dex), by Merck Group (4 mentions)
Epidermal growth factor (egf), by R&D Systems (3 mentions)
Ang 2, by R&D Systems (2 mentions)
Gastrin, by Merck Group (2 mentions)
A83 01, by Bio-Techne (2 mentions)
Hepatocyte growth factor (hgf), by Thermo Fisher Scientific (2 mentions)
Dispase, by Thermo Fisher Scientific (1 mentions)
Collagenase type 2, by Thermo Fisher Scientific (1 mentions)
Insulin, by Sartorius (1 mentions)
Glucagon, by RayBiotech (1 mentions)
L cycloserine, by Merck Group (1 mentions)
Ru486, by Merck Group (1 mentions)
Corticosterone, by Merck Group (1 mentions)
Vegf c, by Thermo Fisher Scientific (1 mentions)
Hepatocyte growth factor (hgf), by R&D Systems (1 mentions)
Vegf a, by Cloud-Clone (1 mentions)
Horse serum, by Sartorius (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Fgf21, by Merck Group (1 mentions)
12 protocols using fgf19
1
Measurement of Serum Angiogenic Factors
2
Canine Hepatic Progenitor Cell Isolation and Differentiation
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Hepatic progenitor cells were isolated from canine liver tissue upon mechanical dissection and subsequent type-II collagenase (Gibco, Landsmeer, the Netherlands) and dispase (Gibco, Landsmeer, the Netherlands) digestion, as described previously [20 (link)]. Expansion medium (EM) conditions, medium changes and split ratio were as described, in detail, in [20 (link)]. Organoid stem cell differentiation towards hepatocytes was induced by the addition of 25 ng/mL BMP7 (Peprotech, Heerhugowaard, the Netherlands) after the last passage in expansion medium. Four days after the last passage, Wnt-conditioned medium, ROCK inhibitor, and Noggin were withdrawn from the medium. Further differentiation was acquired by withdrawal of nicotinamide, R-spondin-1-conditioned medium, and FGF10 six days after the last passage in expansion medium. Finally, BMP7 was continued and 100 ng/mL FGF19 (R&D Systems, Abingdon, UK), 10 µM DAPT (Selleckchem, Huissen, the Netherlands), and 30 µM dexamethasone (Sigma-Aldrich, Zwijndrecht, the Netherlands) were added. This culture was continued for over one week. The lentiviral transduction to supplement COMMD1-deficient organoids with the correct COMMD1 coding sequence was as described previously [20 (link)].
3
Cellular Signaling Modulation Assay
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Cells were treated with the following reagents at the specified concentrations: glucagon 100 nmol/L (RayBiotech, Peachtree Corners, GA; cat# 228-10549-1); corticosterone 1 μmol/L (Sigma-Aldrich, St Louis, MO; 50-22-6); insulin 1 μmol/L (Biological Industries, Beit HaEmek, Israel; 41-975-100); FGF19 80 ng/mL (R & D, Minneapolis, MN; 969-FG); H-89 20 μmol/L (Sigma-Adlrich; B1427); RU-486 2 μmol/L (Sigma-Aldrich; 84371-65-3); L-cycloserine 300 μmol/L (Sigma-Aldrich; 339-72-0); and AOA 1 mmol/L (Cayman Chemical Company, Ann Arbor, MI; 28298).
4
Serum Growth Factor Profiling in Disease
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We evaluated serum VEGF-A, VEGF-C, VEGF-D, ANG-2, HGF, EGF, and FGF-19, levels using commercial enzyme-linked immunosorbent assays, according to the manufacturer’s protocols. (ANG-2, FGF-19, HGF, and EGF: R&D Systems, Minneapolis, MN, USA; VEGF-A: Cloud-Clone Crop, Wuhan, China; VEGF-C, VEGF-D: Thermo Fisher Scientific, Waltham, MA, USA).
We analyzed changes in serum growth factors at baseline, as well as the best overall response and progressive disease (PD) points.
We analyzed changes in serum growth factors at baseline, as well as the best overall response and progressive disease (PD) points.
5
C2C12 Myoblast Differentiation and Treatment
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C2C12 mouse myoblasts (ATCC) were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) containing 4.5 g/L D‐glucose, 10% foetal bovine serum (Gibco) and 1% penicillin‐streptomycin solution (HyClone) in appropriate conditions. When the cells reached 90%‐100% confluence, they were incubated in DMEM containing 2% horse serum (HS, Bioind, Israel) to promote differentiation into myotubes for 5 days. The myotubes were treated with 100 ng/mL FGF19 (R&D) and 0.5 mM PA (Sigma‐Aldrich) for 24 hours. The dissolution method and concentration selection of FGF19 and PA were consistent with our previous study.22
6
Authenticating and Culturing Cell Lines
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Human cancer cell lines originated from the CCLE [18 (link)] were authenticated by single-nucleotide polymorphism analysis and tested for mycoplasma infection using a PCR-based detection technology (RADIL, University of Missouri, Columbia, MO; now IDEXX Laboratories). All cell lines used were directly thawed from the CCLE collection stock and cultured as previously described [18 (link)] except SW1736 (Creative Bioarray). Cell lines were used within 15 passages of thawing and continuously cultured for less than 6 months. All small molecule inhibitors used were synthesized and structurally verified by NMR and LC/MS according to cited references at Novartis except RMC-4550 (Selleck Chemicals), NSC-87877 (Selleck Chemicals), and IIB-08 (Millipore Sigma). EGF and FGF19 were purchased from R&D Systems.
7
Directed Hepatocyte Differentiation from IHCC Organoids
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As shown in Fig. 2a , hepatocyte differentiation using IHCC organoids was conducted as described previously27 (link). IHCC organoids were cultured for 7–10 days in EM additionally supplemented with BMP7 (25 ng/ml). Then, the culture medium was changed to differentiation medium (DM) composed of Advanced DMEM/F12 supplemented with Glutamax, 10 mM HEPES, penicillin/streptomycin, 1 × N2 supplement, 1 × B27 supplement, 50 ng/mL EGF, 50 nM gastrin, 25 ng/ml HGF (Peprotech), 100 ng/ml FGF19 (R&D systems), 10 μM DAPT (Sigma-Aldrich), 25 ng/ml BMP7 (Peprotech), and 30 μM dexamethasone (Sigma-Aldrich). DM was changed every 2–3 days for a period of 12 days.
8
Hepatocyte Differentiation from Organoids
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Differentiation of organoids into hepatocytes was induced as previously described18 (link). Briefly, after amplification organoids were first cultured in EM media supplemented by BMP7 (25 ng/ml) for 7–10 days, then split and plated back in the same media for 2–4 days. Organoids were subsequently switched to the DM differentiation media, composed as follows: AdDMEM/F12 medium (Life Technologies), 1% N2 and 1% B27 (Life Technologies), 50 ng/mL EGF (R&D Systems), 10 nM gastrin (Sigma), 25 ng/mL HGF (Miltenyi Biotec), 100 ng/mL FGF19 (R&D Systems), 500 nM A83.01 (Tocris), 10 μM DAPT (Sigma), 25 ng/mL BMP7 (Peprotech), and 30 μM Dexamethasone (Sigma).
9
Porcine ICO Differentiation Protocol
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Prior to differentiation, porcine ICOs from 4 donors (n = 3 per donor) in the same passage number (p4) were cultured in EM with 25 ng/ml BMP7 (Peprotech) for 5 days. Differentiation medium (DM) was based on advanced DMEM/F12 (Life Technologies) containing 1% (v/v) Glutamax (Life Technologies), 1% (v/v) Penicillin–Streptomycin (Life Technologies), 1% (v/v) HEPES (Life Technologies), 1% (v/v) N2 (Invitrogen), 2% (v/v) B27 without vitamin A (Invitrogen), 1.25 mM N‐acetylcysteine (Sigma‐Aldrich), 5 μM A83‐01 (Tocris Bioscience), 50 ng/mL human epidermal growth factor (EGF, Invitrogen), 0.1 μg/ml human noggin (Peprotech), 0.1 μg/ml FGF19 (R&D Systems, Minneapolis, MN, USA), 10 nM gastrin (Sigma‐Aldrich), 25 ng/ml hepatocyte growth factor (HGF, Peprotech), 30 μM dexamethasone (Sigma‐Aldrich), 10 μM DAPT (y‐secretase inhibitor, Selleckchem), and 25 ng/ml BMP7 (Peprotech), and 1% (v/v) primocin (Gentaur, Kampenhout, Belgium). Differentiation medium was refreshed every 3 days until the end of differentiation culture (Day 5 and 9).
10
Western Blot and qRT-PCR Analysis
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Cells were lysed for protein or RNA extraction, and subjected to western blot or cDNA synthesis and qRT-PCR was as previously described (24 (link)). Antibodies for western blot were as follows: β-Actin (Proteintech,66009-1-Ig), β-Tubulin ( Proteintech, 66240-1-Ig) , FGF19 (R&D System, AF969), FGFR4 (R&D System, MAB6852), FRS2 (Abcam, ab10425), pFRS2 (R&D System, AF5126), goat anti-mouse IgG-HRP (Jackson ImmunoResearch, 115-035-003), goat anti-rabbit IgG-HRP (Jackson ImmunoResearch, 111-035-003), donkey anti-Goat IgG-HRP (Sangon Biotech, D110115) and other antibodies were obtained from the Cell Signaling Technology. Primers for qPCR were as following: CCND1, forward 5′-GTCCTACTTCAAATGTGTGCAG-3′, reverse 5′-GGGATGGTCTCCTTCATCTTAG-3′; GAPDH forward 5′-GGAGCGAGATCCCTCCAAAAT-3′, reverse 5′-GGCTGTTGTCATACTTCTCATGG-3′.
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