Ruxolitinib (CAS 941678–49-5, Cat#11609) and fedratinib (TG101348) were purchased from Cayman Chemical, dissolved in DMSO, and stored as 5 μl stock aliquots: 20 mM Ruxolitinib at −20 °C and 10 mM fedratinib at −80 °C. Recombinant human TNF-α was obtained from R&D Systems (#210-TA). Nuclear extracts were prepared by using the kit from Cayman Chemical (#10009277) following the manufacturer’s guidelines. Protein was quantified by using the Pierce BCA Protein Assay Kit (ThermoScientific Cat#23225). Human factor X (#HCX-0050) and human factor VIIa (HCVIIA-0031) were purchased from Haematologic Technologies. Chromogenic substrate for the determination of factor Xa activity (Chromogenix S-2765, Diapharma) was reconstituted per manufacturer’s instructions and stored at 4 °C. The urokinase type plasminogen activator human chromogenic activity assay kit was purchased from Abcam (Cat#ab8915) and used following the manufacturer’s protocol. HEPES buffered saline [HBS] (20 mM HEPES, pH 7.4, 150 mM NaCl) was used for tissue factor assay with the addition of 3% w/v bovine serum albumin (Fraction V; Fischer). CaCl2 (10 mM, final concentration) was added. For quenching the reactions, EDTA (10 mM final concentration) was added.
Ruxolitinib
Manufactured by Cayman Chemical
Sourced in United States
Ruxolitinib is a lab equipment product manufactured by Cayman Chemical. It is a Janus kinase (JAK) inhibitor that targets the JAK1 and JAK2 enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Stattic, by Cayman Chemical (2 mentions)
Zileuton, by Cayman Chemical (2 mentions)
Imatinib, by Cayman Chemical (2 mentions)
Deferoxamine mesylate salt, by Merck Group (2 mentions)
Ferrostatin 1, by Cayman Chemical (2 mentions)
Ml162, by Cayman Chemical (2 mentions)
Liproxstatin 1, by Cayman Chemical (2 mentions)
Mouse ifnγ 485 mi, by R&D Systems (2 mentions)
Gemcitabine, by Selleck Chemicals (2 mentions)
Erastin, by Cayman Chemical (2 mentions)
Liperfluo, by Dojindo Laboratories (2 mentions)
Anti ifnγ xmg1.2, by Thermo Fisher Scientific (2 mentions)
L buthionine sulfoximine, by Merck Group (2 mentions)
Siinfekl peptide ova 257 264, by Merck Group (2 mentions)
Ml210, by Cayman Chemical (2 mentions)
Jak inhibitor 1, by Cayman Chemical (2 mentions)
Doxorubicin adriamycin hcl, by Selleck Chemicals (2 mentions)
Recombinant human ifnγ 285 if, by R&D Systems (2 mentions)
Bodipy 581 591 c11, by R&D Systems (2 mentions)
Anti tnf α mp6 xt22, by Thermo Fisher Scientific (2 mentions)
32 protocols using ruxolitinib
1
Biochemical Assays for Coagulation Factors
2
Immune Modulation by Nucleic Acids
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It is well known that poly(I:C) stimulates innate immunity such as TLR3.33 Thus, it was used for surrogate viral RNA such as rhinovirus.13 We also used CpG oligonucleotides (CpG–ODN), TLR9 ligand replacement of viral DNA. Both nucleic acid compounds were purchased from Novus Biologicals. IL‐13 and IL‐4 were purchased from PeproTech. IL‐33 was purchased from Wako Pure Chemical. IL‐37 and CC16 (Clara cell secretory protein; a marker of bronchial lung epithelial cells) were purchased from ProSpec and R&D Systems, respectively. BAY 11‐7082 (an inhibitor of nuclear factor kappa light chain (NF‐κB) enhancer of activated B cells was purchased from InvivoGen. Ruxolitinib, stattic (an inhibitor of signal transducer and activator of transcription 334 ), and LY294002 (a phosphatidylinositol kinase‐3 inhibitor) were purchased from Cayman Chemical. The chemotherapy agent, fludarabine, was purchased from Wako Pure Chemical. The corticosteroid, FP, was purchased from Sigma. A type I interferon (IFNs) neutralizing antibody mixture was purchased from PBL Assay Science. Small interfering RNAs (siRNAs) for TLR3, interferon regulatory factor (IRF) 3, RelA (a NF‐κB subunit), JAK1, STAT6, extracellular signal‐regulated kinase (ERK) 1, ERK2, Stealth RNAi siRNA Negative Control Med GC Duplex #2, and Lipofectamine RNA iMAX Reagent were purchased from Invitrogen.
3
SARS-CoV-2 Inhibition by IAV DIPs
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Confluent Calu-3 cells in 96-well plates (~6 × 104 cells/well) were infected with SARS-CoV-2 (2000 pfu per well). At 1 or 24 hpi, we added active or inactive IAV DIPs (DI244 or OP7) at indicated fractions (% v/v) with respect to the cell culture volume of 100 µL. Whenever indicated, we additionally added 0.8 µM ruxolitinib (Cayman Chemical (Ann Arbor, MI, USA), #Cay11609-1) to these wells. Alternatively, remdesivir (MedChem Express (Monmouth Junction, NJ, USAnited States), #HY-104077) or human IFN-β-1A (PBL assay science (Piscataway, NJ, USA), #11415-1) (instead of IAV DIPs) were added at indicated concentrations at 1 hpi. Supernatants were collected at indicated time points for quantification of SARS-CoV-2 titers (plaque assay) and for protein quantification of secreted IFNs using commercially available ELISA kits (see below). In addition, infected cells were lysed using solution RL for subsequent total RNA extraction using the innuPREP RNA Mini Kit 2.0 (Analytik Jena (Jena, Germany), #845-KS-2040050), according to the manufacturer’s instructions, for gene expression analysis via real-time RT-qPCR.
4
Immunofluorescence Analysis of HPV16 Infection
5
Mesenchymal Stem Cells' Response to Cancer Microenvironment
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BM-MSCs were cultured until to 80–90% confluence and serum-starved overnight using DMEM/LG supplemented with 2 mM L-glutamine and 100 U/mL P/S. Then, the BM-MSCs were treated with MDA CM, MCF7 CM, or CON CM for the indicated times. For JAK and ROS inhibition, the cells were pretreated with ruxolitinib (Cayman Chemical, Ann Arbor, MI, USA) and N-acetylcysteine (NAC; Sigma-Aldrich), respectively, for 30 min prior to the CM treatment. BM-MSCs were rinsed twice with ice-cold PBS and lysed using 2× SDS buffer (100 mM Tris-HCl, pH 6.8, 20% (v/v) glycerol, 2% (v/v) sodium dodecyl sulfate (SDS), 0.001% (w/v) bromophenol blue, and 10% (v/v) β-mercaptoethanol) at 25 °C for 5 min. The cell lysates were collected by scrapping and denatured by heating at 95 °C for 5 min. Western blot analysis was performed with the following primary antibodies: anti-HIF-1α (1:800-1:1000; Cell Signaling Technology, Danvers, MA, USA), anti-phospho-Stat3 (pStat3; 1:500–1:800; Cell Signaling Technology), anti-Stat3 (1:1000; Cell Signaling Technology), and α-tubulin (1:30000–1:50000, Sigma-Aldrich). Densitometry of the bands obtained was performed using the ImageJ software (NIH, Bethesda, MD, USA).
6
hCEC Viability, Inflammation, Barrier Assay
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SB431542 (SB; Cayman Chemical, Ann Arbor, MI, US) and ruxolitinib (ru; Cayman Chemical) were dissolved in dimethyl sulfoxide (Wako) as stock solutions. hCECs were treated with SB431542 (1, 10, and 30 µM) or ruxolitinib (0.3, 3, and 30 µM). Next, hCECs were analysed for viability, inflammation, and barrier function.
7
Cytotoxicity Assay of Small Molecules
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Rhabdomyosarcoma cell lines Rh30 and Rh6 were seeded in 384-well plates at 2500 cells per well, in 30 µL of media, using a BioTek MultiFlo dispenser. Cultures were grown overnight and then treated with drugs FGF401 (23029), PP1 (14244), Ruxolitinib (11609), and Stattic (14590) that were all purchased from Cayman Chemical. All of the pharmaceuticals were administered at the following concentrations using a D300e digital dispenser (Tecan Trading AG): 0.01, 0.0316, 0.1, 0.316, 1, 3.16, and 10 µM. After 72 h of drug treatment, cultures were treated with an equal volume of CellTiter-Glo reagent (Promega 2020-01-29). Luminescence was measured in a Biotek Instruments Inc. plate reader. The data was then analyzed using Prism GraphPad software.
8
Zileuton and Ruxolitinib Treatment for PV Mice
9
Mast Cell Activation Assay with IgE and Inhibitors
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Mast cells were incubated overnight with 100 ng/ml murine monoclonal anti-DNP IgE, clone SPE-7 (D8406, RRID:AB_259249 , Millipore-Sigma, St. Louis, MO) at 37 C. The next day, cells were centrifuged and washed twice with fresh media to remove unbound IgE and cells were replated in fresh media with 10 ng/ml IL-3. Stimulation was to the relevant wells added in the form of 10 ng/ml IL-33 and/or 100 ng/ml DNP-BSA for 24 h at 37 C, then cells were centrifuged and supernatant was collected. For the drug inhibition assays, ibrutinib, ruxolitinib, imatinib, and fingolimod were purchased from Cayman Chemical (936563-96-1, 941678-49-5, 220127-57-1, 162359-55-9, Ann Arbor, MI) and reconstituted per the manufacturer's instructions. Drug stocks were diluted in media and incubated with mast cells after the overnight IgE incubation step. Cells were treated for 1 h at 37 C, then washed out with fresh media twice. Cells were then stimulated as per above.
10
Inflammatory Response Regulation in hDPCs
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All methods and experiments were approved by the Ethical Committee of Tokyo Medical and Dental University and carried out in accordance with the relevant Ethical Guidelines and Regulations for Clinical Studies (Reference number D2014-039). All participants provided informed consent in accordance with the Ethical Guidelines and Regulations for Clinical Studies. hDPCs were isolated from dental pulp tissue of human third molars and cultured in α minimum essential medium (αΜΕΜ; FUJIFILM Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin/streptomycin (FUJIFILM Wako Pure Chemical) under a standard condition (37 °C, 5% CO2). The culture medium was changed at 3-day intervals, and cells from passages 2–7 were used. To provide an inflammatory stimulus to hDPCs, LPS (Escherichia coli O111:B4, 100 ng/mL; Sigma-Aldrich, St. Louis, MO, USA) was applied. BAY 11-7085 (CAS Number: 196309-76-9, 1 μM; Cayman Chemical, Ann Arbor, MI, USA), a specific and potent NF-κB signaling inhibitor [44 (link),45 (link)], was used to block the NF-κB signaling pathway. Ruxolitinib (CAS Number: 941678-49-5, 10 μM; Cayman Chemical), a specific and potent JAK1/2 inhibitor [46 (link),47 (link)], was used to block the JAK/STAT3 signaling pathway.
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