Dorsal root ganglia (DRG) neurons were isolated from mice.3 (link) Cells were washed and incubated with Hank's Balanced Salt Solution (HBSS)+Ca2+, Fluo-4-AM (Invitrogen) and 20% pluronic F-127 (Invitrogen) during 30 min at 37°C followed by 30 min at room temperature before imaging.9 (link) Supernatants from apical or basal compartments of Caco-2 cells culture, stimulated or not by LPS (Escherichia coli serotype K235, ATCC13027), were pre-incubated with the serine protease inhibitor FUT-175 (Futhan) (50 μg/mL) or the trypsin inhibitor leupeptin (100 μM) (all from Calbiochem) for 15 min. Additional recordings were made from DRG neurons after addition of trypsin-3 (10 nM, R&D) or its vehicle (HBSS+0.025% Brij35) in the presence or absence of specific PAR1 (SCH79797, Tocris Bio-Techne, UK) or PAR4 (ML-354, Tocris Bio-Techne) antagonists (all at 10 µM for 5 min). Neurons were identified by addition of high K+ (50 mM) solution at the end of the recording. Fluo-4 was excited at 475 nm, and fluorescence emission was collected at 490/515 nm. Changes in [Ca2+]i are reflected by Fluo-4 fluorescence intensity.
Sch79797
Manufactured by Bio-Techne
Sourced in United Kingdom
SCH79797 is a small molecule inhibitor that acts as a thrombin receptor (protease-activated receptor-1, PAR-1) antagonist. It is used in research applications to study the role of thrombin receptor signaling in various biological processes.
Automatically generated - may contain errors
Lab products found in correlation
Thrombin, by Merck Group (2 mentions)
Dabigatran, by Boehringer Ingelheim (2 mentions)
Pluronic f 127, by Thermo Fisher Scientific (1 mentions)
Ml 354, by Bio-Techne (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Aypgkf, by AnaSpec (1 mentions)
Irbesartan, by Merck Group (1 mentions)
Anti ha tag antibody ab9110, by Abcam (1 mentions)
Ang 2, by Merck Group (1 mentions)
Pf573228, by Bio-Techne (1 mentions)
2 acetylphenothiazine ml171, by Merck Group (1 mentions)
A23187, by Merck Group (1 mentions)
Fibronectin, by Abcam (1 mentions)
Tfllr nh2, by Bio-Techne (1 mentions)
Anti abcg2, by GeneTex (1 mentions)
Mrp 1, by GeneTex (1 mentions)
Anti gapdh, by Novus Biologicals (1 mentions)
Anti e cadherin, by Abcam (1 mentions)
Hirudin, by Merck Group (1 mentions)
Fibronectin, by Merck Group (1 mentions)
16 protocols using sch79797
1
Calcium Imaging of DRG Neurons
2
Cyr61 Regulation by PAR Agonists
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Cells were treated with 111 µM of PAR-1 or -4 peptide agonists (TFLLR or AYPGKF, AnaSpec, Fremont, CA), corresponding scrambled PAR-1 or -4 peptides (RLLFT or YAPGKF, Tocris Bioscience, Minneapolis, MN), 1.0 U/ml thrombin, or cell naïve medium for 1 h. Lysates were prepared, and the media collected, clarified, and concentrated. Then, the proteins were separated by SDS-PAGE and analyzed using western blot data generated using the H-78 antibody specific for the linker region between the vWF and TSP-1 domains of Cyr61. For PAR-1 inhibition experiments, cells were first pretreated with or without the PAR-1 antagonist, SCH 79797 (Tocris Bioscience), using a concentration of 25 nM for fibroblasts and 50 nM for myofibroblasts for 30 min. Then, either an additional serum-free medium or a medium containing thrombin at a final concentration of 1.0 U/ml was added to the cultures. Cells were further incubated with the combination treatment for 3 h. Lysates and supernates were collected, prepared, and analyzed for Cyr61 using western blot data generated using the H-78 antibody.
3
Transient Protein Expression in HEK293 Cells
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The following human protein-coding plasmids were used for the transient expression in HEK293 cells: PAR1was purchased from cDNA Resource Center (Bloomsberg, PA, USA), PAR1-Rluc was used as previously described (Ayoub et al., 2010 (link)), HA-AT1R was kindly provided by Reiter E. (INRA, Nouzilly, France), AT1R-Rluc was a gift from Laporte, S. (McGill University, Montréal, Canada), yPET-β-arrestin 2 was kindly provided by Scott, M. (Cochin Institute, Paris, France), Grb2-Rluc and Grb2-Venus were kindly given by Prof. Pfleger KD. (Harry Perkins Institute of Medical Research and UWA, Perth, Australia), and Venus-Gαq, Venus-Kras, and Venus-Rabs that were generously shared by Lambert, N. (Augusta University, GA, USA). AngII, thrombin, and irbesartan were from Sigma (St Louis, MO, USA). SCH79797 was from Tocris (Tocris, Ellisville, MO). The IP1 kit was purchased from Cisbio Bioassays (PerkinElmer, Codelet, France) and the anti-HA tag antibody (ab9110) was purchased from abcam (abcam, MA, USA).
4
Platelet activation and inhibition assays
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Anti-GPIbα (AK2) and anti-VWF (5D2) murine monoclonal antibodies have been previously described [26] (link), [27] (link); the irrelevant isotype IgG2 was from BD Pharmingen (Oxford, UK). Rat anti-mouse GPIbα (Xia.G5) IgG2B and rat IgG2B isotype (both FITC conjugated) were obtained from Emfret (Würzburg, Germany). Cross-linked collagen related peptide (CRP) was obtained from Prof. Richard Farndale (Department of Biochemistry, Cambridge University, UK). The protein kinase C activator, phorbol myristoyl acetate (PMA), and the calcium ionophore, A23187, were from Sigma Aldrich (St. Louis, MO, USA). Thrombin was from Calbiochem (UK). PAR1 (PAR1-AP, SFLLRN-NH2) and PAR4 (PAR4-AP, AYPGKF-NH2) agonists were from Abgent Europe (Oxfordshire, UK). PAR4 antagonist, tcY-NH2, PAR1 antagonist, SCH79797, and PF-573228 (hereafter referred to as PF-228) were from Tocris Bioscience (R&D Systems Europe, UK). ML171 (2-acetylphenothiazine) and BMS200261 were purchased from Sigma Aldrich (St. Louis, MO, USA). Nk protease (a GPIbα-specific cleavage enzyme from the venom of cobra Naja kaouthia) was purified as previously described [28] (link).
5
Selective PAR1 Antagonists and Dabigatran
6
Regulation of YAP1 by PAR1 Signaling
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An antibody against PAR1 was purchased from BECKMAN COULTER. Anti-E-cadherin, fibronectin, YAP1, phospho-YAP1 (pYAP1), Lats1 and phospo-Lats1 (pLats1) were purchased from Abcam. Anti-ABCG2, -MRP1, and -P-glycoprotein (P-gp) were purchased from GeneTex. Anti- ribophorin II (RPN2) was purchased from OriGene. Anti-GAPDH was from IMGENEX. The selective PAR1 agonist TFLLR-NH2 and PAR1 antagonist SCH79797 was purchased from Tocris Bioscience. We used TFLLR-NH2 (EC50: 1.9 μM) at the 20 μM, and SCH79797 (IC50: 70 nM) at the 70 nM. C3 transferase and Y27932 were purchased from Cytoskeleton. We used C3 at the 2 μg/ml and Y27632 at 10 μM. Small interfering RNA (siRNA) directed against PAR1 (5′-AAGGCUACUAUGCCUACUACU-3′) was synthesized by TOYOBO, and siRNA directed against YAP1 (5′-GGUCAGAGAUACUUCUUA-3′) and Snail (5′- CCACAGAAAUGGCCAUGGGAAGGCCUC-3′) were synthesized by Sigma. Control siRNA is a scrambled sequence with no homology in the human genome (Qiagen) is listed as follows: Scrambled, UUCUCCGAACGUGUCACGUdTdT.
7
Farnesylthiosalicylic Acid and PAR1 Antagonist in SOD1 Mice
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Treatments were administrated five times a week. FTS (S-trans-trans-farnesylthiosalicylic acid) was administrated orally (volume of 100 µL) in low and high dosages (25, 40 and 60 mg/kg, respectively) utilizing an appropriate gavage. TLCK (N-Tosyl-Lys-chloromethylketone) was used at a dose of 4.4 mg/kg and PAR1 antagonist (SCH-79797, Tocris, Bristol, UK) was used at a dose of 25 μg/kg, both were administered by intraperitoneal injections (Figure 4 ).
Animal sample size used in the study: thrombin activity: SOD1 n = 11, healthy control n = 7. PAR1 levels: healthy control n = 5, SOD1 n = 4. Immunostaining: SOD1 n = 3, healthy control n = 4. Weight: healthy control n = 6, SOD1 n = 22, TLCK n = 21, PAR1 antagonist n = 14, FTS-40 n = 21, FTS-25 n = 14. Rotarod: SOD1 n = 21, TLCK n = 20, PAR1 antagonist n = 14, FTS-40 n = 5, FTS-25 n = 13, Healthy control n = 5. Survival: SOD1 n = 22, PAR1 antagonist n = 13, TLCK n = 19, FTS-25 n = 14, FTS-40 n = 21, FTS-60 n = 19, healthy control n = 6.
Animal sample size used in the study: thrombin activity: SOD1 n = 11, healthy control n = 7. PAR1 levels: healthy control n = 5, SOD1 n = 4. Immunostaining: SOD1 n = 3, healthy control n = 4. Weight: healthy control n = 6, SOD1 n = 22, TLCK n = 21, PAR1 antagonist n = 14, FTS-40 n = 21, FTS-25 n = 14. Rotarod: SOD1 n = 21, TLCK n = 20, PAR1 antagonist n = 14, FTS-40 n = 5, FTS-25 n = 13, Healthy control n = 5. Survival: SOD1 n = 22, PAR1 antagonist n = 13, TLCK n = 19, FTS-25 n = 14, FTS-40 n = 21, FTS-60 n = 19, healthy control n = 6.
8
Investigating Protease-Activated Receptor Signaling
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PAR1 antibodies WEDE-15 and ATAP-2 were purchased from Beckman coulter (Fullerton, CA, USA) and Santa Cruz Biotechnologies (Santa Cruz, CA, USA), respectively. PAR2 antibody SAM-11 was purchased from Santa Cruz Biotechnologies. PAR1 agonist H-Thr-Phe-Leu-Leu-Arg-NH2 (TFLLR) and antagonist Mercaptopropionyl-Phe-Cha-Arg-Lys-Pro-Asn-Asp-Lys-NH2 (Mpr-NH2), PAR2 antagonist H-Phe-Ser-Leu-Leu-Arg-Tyr-NH2 (FSLLRY), and PAR4 antagonist trans-Cinnamoyl-Tyr-Pro-Gly-Lys-Phe-NH2 (tcY-NH2) were purchased from Peptides International (Louisville, KY). Anti-rat CD18 (clone WT3) and anti-rat CD11a (clone WT1) were from AbD Serotec (Kidlington, UK). Polyclonal Abs specific for phosphorylated forms of eNOS, p38, JNK, ERK1/2 (eNOS Ser1177, Erk1/2 Thr202/Tyr204, p38 Thr180/Tyr182 and SAPK/JNK Thr183/Tyr185) were from Cell Signaling Technology (Beverly, MA, USA); the anti-phosphotyrosine (4G10) was from Upstate Biotechnology, Millipore (Billerica, MA, USA), and the monoclonal antibody for tubulin was from Sigma-Aldrich (St Louis, MO, USA). Thrombin, Hirudin, BAPTA-AM, L-NAME (NG-nitro-L-arginine methyl ester), collagen IV and fibronectin were from Sigma-Aldrich. Compound C and antiThrombin III were from Merck Biosciences (Nottingham, UK). SCH79797 was from Tocris (Bristol, UK).
9
SARS-CoV-2 Infection of Platelets and Monocytes
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SARS-CoV-2 was originally isolated from nasopharyngeal swabs of a confirmed case from Rio de Janeiro/Brazil (GenBank accession no. MT710714). The virus was amplified for 2 to 4 days in Vero E6 cell cultures in high-glucose Dulbecco Modified Eagle’s Medium supplemented with 2% fetal bovine serum at 37°C in 5% CO2 atmosphere. Virus titers were determined by the tissue culture infectious dose at 50% and the virus stocks kept in −80°C freezers until use. All procedures involving SARS-CoV-2 culture were performed in a biosafety level 3 facility. Platelets (2 × 107) and monocytes (2 × 105) were infected with SARS-CoV-2 separately or in combination (multiplicity of infection = 0.01 virus per monocyte). In selected experiments, platelet-monocyte cocultures were infected in the presence of abciximab, anti-TF antibodies (clone 10H10 or 5G9), or isotype-matched IgG (50 µg/mL), or the PAR-1 inhibitor SCH79797 (5 µM, Tocris 1592), PAR-2 inhibitor AZ3451 (10 µM, Sigma SML2050), or DMSO (vehicle). After 12 hours of infection, supernatants were harvested and stored for future analysis, and cells were fixed with 4% paraformaldehyde for flow cytometry analysis as described in supplemental Material.
10
Selective PAR1 Inhibition and Electrophysiology
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SCH79797 [IC50 = 70 nM; Tocris Bioscience, UK] was used to block PAR1. Tetrodotoxin (TTX) and D(-)-2-amino-5-phosphonovaleric acid (D-AP5; both from Tocris Bioscience, UK) were used for electrophysiological recordings. Handling and disposal of all drugs carried out in accordance to German and University regulations.
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