LPS from E. coli and P. gingivalis was dissolved in 1× phosphate-buffered saline at a final concentration of 10 mg/mL and stored at 4 °C. The hPDLCs were treated with different concentrations of LPS (0, 10, 20, 50, and 100 μg/mL) from P. gingivalis and E. coli freshly diluted in culture medium. The adenylyl cyclase inhibitor SQ22536 (Sigma-Aldrich, St. Louis, MO, USA) was used to block the activity of adenylyl cyclase. SQ22536 was dissolved in dimethyl sulfoxide (DMSO), and the cells were treated with a dose of 100 μmol/L. Forskolin (50 μmol/L, Sigma-Aldrich, St. Louis, MO, USA), an adenylyl cyclase activator, was dissolved in DMSO.
Sq22536
Manufactured by Merck Group
Sourced in United States, Germany, United Kingdom
SQ22536 is a laboratory product manufactured by Merck Group. It is a chemical compound that functions as an adenylyl cyclase inhibitor. This compound is commonly used in scientific research applications.
Automatically generated - may contain errors
Lab products found in correlation
Forskolin, by Merck Group (18 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (5 mentions)
3 isobutyl 1 methyl xanthine (ibmx), by Merck Group (5 mentions)
Rolipram, by Merck Group (4 mentions)
U73122, by Merck Group (4 mentions)
Calyculin a, by Merck Group (3 mentions)
Kt5720, by Merck Group (3 mentions)
Esi 09, by Merck Group (3 mentions)
Alloxan, by Merck Group (2 mentions)
Heparin, by Merck Group (2 mentions)
Ly294002, by Merck Group (2 mentions)
Phosphate buffered saline (pbs), by Welgene (2 mentions)
Pancuronium bromide, by Merck Group (2 mentions)
Methacholine, by Merck Group (2 mentions)
Carbachol, by Merck Group (2 mentions)
Ethylene glycol tetraacetic acid (egta), by Merck Group (2 mentions)
Des his1 glu9 glucagon amide, by Merck Group (2 mentions)
Penicillin, by Merck Group (2 mentions)
Lipopolysaccharides (lps), by Merck Group (2 mentions)
Glucagon, by Merck Group (2 mentions)
61 protocols using sq22536
1
Investigating Lipopolysaccharide-Induced Responses in hPDLCs
2
Pancreatic Cancer Cell Culture Protocol
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The human pancreatic cancer cell lines SW1990 and BxPC-3 were obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium containing 10% foetal bovine serum (Thermo Fisher Scientific) and 1% penicillin-streptomycin at 37 °C with 5% CO2. Adenosine (018–10,492) was purchased from Wako (Osaka, Japan) and 8-CPT (#C0735), DMPX (#D134), alloxan (#A7413), MRS1523 (#M1809), EHNA (#E114), forskolin (#F6886), SQ22536 (#S153), H89 (#B1427), and dipyridamole (#D9766) were purchased from Sigma (Shanghai, China); HPBCD (#A600388) was from Sangon Biotech (Shanghai, China); and GSK690693 (#HY-10249) was from MCE (New Jersey, USA). For in vivo studies, Adenosine and GSK690693 were dissolved in 10% 2-hydroxypropyl-β-cyclodextrin (Sangon Biotech, Shanghai, China).
3
Nobiletin Modulates Cell Signaling Pathways
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Nobiletin (≥97%), collagen (type I), 5,5-dimethyl-1-pyrroline N-oxide (DMPO), SQ22536 (inhibitor of adenylate cyclase), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; inhibitor of guanylate cyclase), heparin, PGE1, nitroglycerin (NTG), H89 (inhibitor of PKA), KT583 (PKG inhibitor), LY294002 (Akt inhibitor), Ro318220 (PKC inhibitor), DPI (NOX inhibitor), Bay11-7082 (NF-κB inhibitor), PD98059 (ERK2 inhibitor), SB203580 (p38 MAPK inhibitor, SP600125 (JNK inhibitor) and N-acetylcysteine (NAC) were all purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-VASP (Cat. no. GTX132176) and anti-VASP (phospho Ser157; Cat. no. GTX32362) polyclonal antibodies (pAbs) were purchased both from GeneTex, Inc. (Irvine, CA, USA). The anti-α-tubulin monoclonal antibody (mAb; Cat. no. MS-581-P0) was purchased from NeoMarkers (Fremont, CA, USA). Hybond-P polyvinylidene difluoride (PVDF) membranes, an enhanced chemiluminescence western blotting detection reagent, the horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG; Cat. no. RPN4301), and the sheep anti-mouse IgG (Cat. no. RPN4201) were all purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). Nobiletin was dissolved in 0.5% dimethyl sulfoxide (DMSO) and stored at 4°C.
4
Adipocyte Differentiation Assay with Filbertone
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3T3-L1 preadipocytes were obtained from American Type Culture Collection (ATCC Manassas, VA, USA) and cultured in a humidified atmosphere with 5% CO2 at 37 °C. The cells were differentiated into adipocytes as previously reported [14 (link)]. Briefly, 3T3-L1 preadipocytes were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% bovine calf serum (Gibco life technologies, Carlsbad, CA, USA) and 1% antibiotic–antimycotic solution (Gibco life technologies). For the differentiation assay, preadipocytes were seeded in six-well plates and cultured until two days post-confluence. After the 3T3-L1 cells became confluent, the medium was replaced with DMEM consisting of 10% of fetal calf serum (FBS; Gibco-BRL), 1% of antibiotic–antimycotic solution, 10 μg/mL bovine insulin, 1 μM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), and 0.5 mM isobutylmethylxanthine (Sigma-Aldrich) with or without filbertone (Sigma-Aldrich, St. Louis, MO, USA) and SQ22,536 (an adenylate cyclases (ADCY) inhibitor, Sigma-Aldrich, St. Louis, MO, USA). The cells were then maintained in DMEM containing 10% of FBS with 10 μg/mL insulin for another two days, followed by culturing with DMEM containing 10% of FBS for an additional four days with or without filbertone and SQ22,536.
5
Protein Phosphorylation Regulation in Cells
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Protein phosphatase 1 and 2A inhibitor (Calyculin A), protein kinase A inhibitor (H-89), and adenylyl cyclase inhibitor (SQ22,536) were purchased from Sigma-Aldrich. The following primary antibodies were used for western blotting: myosin light chain 2 antibody (#3672; Cell Signaling Technology, MA, USA), phospho-myosin light chain 2 (Ser19) antibody (#3671; Cell Signaling Technology), phospho-myosin light chain 2 (Thr18/Ser19) antibody (#3674; Cell Signaling Technology), myosin light chain 2 antibody (#3672; Cell Signaling Technology), GAPDH antibody (#AM4300; Ambion, TX, USA), MYPT1 antibody (#2634; Cell Signaling Technology), phospho-MYPT1 (Thr696) antibody (#5163; Cell Signaling Technology), phospho-MYPT1 (Thr853) antibody (#4563; Cell Signaling Technology), RhoA antibody (#ARH03; Cytoskeleton, CO, USA), and phospho-RhoA (Ser188) antibody (#AB41435; Abcam, Cambridge, UK). MRLC stained by phospho-myosin light chain 2 (Ser19) antibody is denoted as 1P-MRLC, and MRLC stained by phospho-myosin light chain 2 (Thr18/Ser19) antibody is denoted as 2P-MRLC.
6
Melanin Content Quantification in B16F10 Cells
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B16F10 cells (1 × 105 cells/well) were cultured for 48 h in a 12-well plate with either vehicle (DMSO; Sigma, St. Louis, MO, USA) or various concentrations (50–200 µM) of carvone (Sigma; purity 97%). If needed, the cells were pretreated with either vehicle (DMSO) or 50 µM SQ22536 (cAMP inhibitor; Sigma) for 30 min before the carvone treatment. The cells were harvested by trypsinization followed by washing with phosphate-buffered saline (PBS; WelGENE, Daegu, Korea). Each cell sample was resuspended in 400 μL of 1 N NaOH containing 10% of DMSO and heated at 70 °C for 2 h. Melanin amounts were estimated by means of absorbance at 480 nm on a microplate reader (M200; Tecan, Männedorf, Switzerland). Throughout all experiments, the final DMSO concentration was less than 0.05%.
7
Compound Characterization and Stability of JME-173
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Sodium chloride, potassium chloride, potassium dihydrogen phosphate, sodium hydrogen carbonate, magnesium sulfate heptahydrate, and calcium chloride dehydrate were purchased from Merck (Darmstadt, Germany). Glucose, EGTA, carbachol, methacholine, theophylline, mexiletine, propranolol, SQ22,536, lipopolysaccharide (LPS), sodium pentobarbital, pancuronium bromide, sodium orthovanadate, and tetraethylammonium (TEA) were purchased from Sigma-Aldrich (St. Louis, MO). Isoflurane was obtained from Cristália (São Paulo, Brazil). JME-173 was synthesized and provided by the Laboratory of Organic Chemistry (Farmanguinhos, FIOCRUZ, RJ, Brazil). GCMS spectrum revealed that JME-173 is a 100% pure compound as also attested by the 1H NMR analysis and by its melting point 220–222°C. As predicted by the ChemAxon program (Version 19.22.0), the aqueous solubility at pH 6.4, 6.8, and 7.2 of JME-173 were 127.0, 50.6, and 20.2 mg/ml, respectively. There was no observed evidence of JME-173 degradation following long-lasting exposure to blood samples, storage at room temperature or −70°C, or after the repeated freeze and thaw cycles, pointing out the excellent physical stability of this compound (Pinto at al., under review in the Eur. J. Pharmacol.). All solutions were freshly prepared in distilled water and protected from light.
8
Dopamine Signaling Assay Protocol
9
Autophagy Modulation in Cell Imaging
10
Neutrophil Activation Assay Protocol
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Alloxan, Glucagon, Des-his1-[Glu9]-Glucagon amide, Rolipram, Percoll, RPMI medium, Hank’s balanced salt solution without Ca+2 or Mg+2 (HBSS), mouse CXCL1/KC, human CXCL8/IL-8, N-formyl methionyl-leucyl-phenylalanine (fMLP), H-89, SQ 22536, DMSO, LPS, Penicillin, Zymosan A and Streptomycin were purchased from Sigma Chemical Co (St. Louis, MO, USA). Ficoll-Paque PLUS (density 1.077 g/mL) was purchased from GE Healthcare Bio-Sciences (Pittsburgh, PA, USA), platelet-activating factor (PAF) was purchased from Calbiochem (San Diego, CA, USA), and fetal bovine serum (FBS) was purchased from Gibco (Albuquerque, NM, USA). Meropenem was purchased from ABL – Antibióticos do Brasil (São Paulo, SP, Brazil) and Luminol was donated by Prof. Margareth Queiroz from Oswaldo Cruz Institute/Fiocruz (Rio de Janeiro, RJ, Brazil). All the solutions were freshly prepared immediately before use.
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