A wound-healing assay was used to analyze the migration of human atrial fibroblasts treated with or without Klotho in different concentrations (10 pM and 100 pM, 48 h, R&D Systems, Minneapolis, MN, USA) 6 h after a cell monolayer in a six-well plate was craped with a P200 pipette tip. To explore the role of TRPC6 and intracellular signaling underlying the effects of Klotho in fibroblasts activity, we evaluated the migration in control, Klotho 100 pM-treated, BI-749327 1 μM (selective TRPC6 inhibitor, MedChemExpress, Monmouth Junction, NJ, USA)-treated, combined Klotho and BI-749327-treated, U73122 1 μM (phospholipase C inhibitor, Abcam, Cambridge, UK)-treated and combined Klotho and U73122-treated human atrial fibroblasts for 48 h. Each gap area was assessed using the ImageJ software (National Institutes of Health, Bethesda, Maryland, USA). The net migration areas after 6 h were subtracted from that at the time of the initial scratches.
U73122
Manufactured by Abcam
Sourced in United Kingdom
U73122 is a phosphoinositide-specific phospholipase C (PLC) inhibitor. It functions by blocking the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG).
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (2 mentions)
Bi 749327, by MedChemExpress (1 mentions)
Sulpiride, by Bio-Techne (1 mentions)
Sulforhodamine 101, by Merck Group (1 mentions)
Dopamine hydrochloride, by Bio-Techne (1 mentions)
Nomifensine, by Bio-Techne (1 mentions)
Strychnine, by Merck Group (1 mentions)
Sch23390, by Bio-Techne (1 mentions)
Chelerythrine, by Bio-Techne (1 mentions)
G 6976, by Bio-Techne (1 mentions)
Akt inhibitor 4, by Merck Group (1 mentions)
Stem cell factor (scf), by Thermo Fisher Scientific (1 mentions)
Anti dnp ige, by Merck Group (1 mentions)
Interleukin 3 (il 3), by Thermo Fisher Scientific (1 mentions)
Ly294002 ab120243, by Abcam (1 mentions)
Dinitrophenyl human serum albumin dnp hsa, by Merck Group (1 mentions)
4 nitrophenyl n acetyl β d glucosamide, by Merck Group (1 mentions)
Lamin b1, by Santa Cruz Biotechnology (1 mentions)
O phthaldialdehyde, by Merck Group (1 mentions)
8 protocols using u73122
1
Klotho Modulates Fibroblast Migration via TRPC6 Signaling
2
Neurochemical Receptor Assay Protocols
3
Mast Cell Activation Assay
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Dinitrophenyl-human serum albumin (DNP-HSA), anti-DNP-IgE, o-phthaldialdehyde, and 4-nitrophenyl N-acetyl-β-d -glucosamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). The protease inhibitor cocktail and phosphatase inhibitor cocktail were purchased from Roche (Mannheim, Germany). IL-3 and stem cell factor (SCF) were purchased from PeproTech (EC, London, UK). The signaling inhibitors, PP2 and Akt inhibitor IV were purchased from Sigma-Aldrich, and U 73122 and LY 294002 (ab120243) were from Abcam (Cambridge, UK). The antibodies for anti-IRF3 and p-S396-IRF3 were obtained from Cell Signaling (Beverly, MA, USA), lamin B1 was obtained from Santa-Cruz Biotechnology (Dallas, TX, USA), and anti-HDC was purchased from Invitrogen (Waltham, MA, USA).
4
PCP and Receptor Modulators Protocol
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Phencyclidine (PCP; Sigma-Aldrich, Gillingham, UK; dissolved in 0.9% saline solution); VU0360172 (N-cyclobutyl-6-((3-fluorophenyl)ethynyl)picolin-amide; Tocris (Bio-Techne), Pittsburgh, USA; stock 10 mM in DMSO); VU0409551(4-fluorophenyl)(2-(phenoxymethyl)-6,7-dihydrooxazolo[5,4-c]pyridin-5(4H)-yl)methanone; Tocris (Bio-Techne), Pittsburgh, USA; stock 10 mM in DMSO); Kainic acid (Hello Bio, Bristol, UK; stock 0.25 mM, in dH2O); Carbachol (Carbamoylcholine chloride; Sigma-Aldrich, Gillingham, UK; stock 50 mM in dH2O); MTEP (3-((2-Methyl-4-thiazolyl)ethynyl)pyridine; Hello Bio, Bristol, UK; stock 200 µM in DMSO); Go6983 (Hello Bio, Bristol, UK; stock 10 µM in DMSO); U73122 (Abcam, Cambridge, UK; stock 10 mM in DMSO).
5
Culturing and Treating Cardiac Fibroblasts
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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.
6
Monocyte-Mediated Coagulation Regulation
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Plates were coated with DPBS or FHR-1 (5 μg mL−1), and 1 × 105 monocytes were added in CM containing 10% NHS with or without addition of the PLC inhibitor U73122 (10 μM, Abcam) or the NF-κB inhibitor BAY11-785 (30 μM, Sigma-Aldrich). After incubation for 20 h at 37 °C in an atmosphere containing 5% CO2, the cells were washed twice with DPBS, followed by the addition of Factor VIIa substrate (0,3 mg mL−1, Sigma-Aldrich) in DPS containing 20% NHS and Mg2+/Ca2+ (Biowest). After 5 min, absorbance was measured at 405 nm. For EMR2 blocking, an anti-EMR2 antibody (10 μg mL−1, AF4894, R&D Systems) was added, and the cells were incubated for 5 h at 37 °C. The cells were washed twice, followed by the addition of Factor VIIa substrate (0.2 mg mL−1, Sigma-Aldrich) in DPBS containing 10% NHS and Mg2+/Ca2+ (Biowest). After 85 min, absorbance was measured at 405 nm.
7
Monocyte-mediated TF Expression Regulation
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High-binding ELISA plates (Sarstedt) were coated with 50 µL/well each of FHR-1 or BSA (5 µg mL−1 each), washed, and incubated with 3 × 105 monocytes/well for 20 h at 37 °C and 5% CO2. NF-κB was inhibited with BAY11-7085 (30 µM, Sigma-Aldrich), and PLC with U73122 (10 µM, Abcam). Cells were harvested with trypsin/EDTA and incubated with sheep anti-human TF (1:100, Hematologic Technologies), followed by incubation with Alexa Fluor 647-conjugated donkey anti-sheep polyclonal IgG (1:2000, ab150179, Abcam) and the eFluor 780 viability dye (1:10,000, eBioscience), each for 20 min at 4 °C in DPBS containing 1% BSA. Between steps, the cells were washed twice with DPBS containing 1% BSA, and fluorescence was measured via flow cytometry. Cells were gated according to forward scattering properties and viability dye uptake.
8
Leukemia Cell Line Culture and Signaling
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The human leukemia cell lines Nalm-6 and Reh (DSMZ®, Deutschland) were cultured in RPMI 1640 medium containing 10% fetal bovine serum, 2 mM of L-glutamine with 5000 UI/L penicillin and 50 mg/L streptomycin (Eurobio®, France), and maintained at 37°C in a 5% CO2 humidified atmosphere. Fura-2/AM, Rhod-2/AM, Bapta-AM, U73122, SKF96365 and thapsigargin were purchased from Abcam Biochemicals, France. Poly-D-lysine, dimethylsulfoxide (DMSO), 2APB and dexamethasone were purchased from Sigma Aldrich, France. PD98059 was purchased from Euromedex, France. Antibodies to total (Erk1/2) and phosphorylated-Erk1/2 (pErk1/2) were obtained from Cell signaling Inc, France.
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