The main reagents used in this experiment include PF-543 citrate (SPHK1 inhibitor; MedChemExpress, HY-15425A, Monmouth Junction, NJ, USA), PD98059 (MEK1/2 inhibitor; MedChemExpress, HY-12028, Monmouth Junction, NJ, USA), S1P (Cayman chemical, 9002921, Ann Arbor, MI, USA), TY-52156 (S1PR3 antagonist; Cayman chemical, 19119, Ann Arbor, MI, USA), wortmannin (PI3K/Akt antagonist; Cayman chemical, 10010591, Ann Arbor, MI, USA). The specific primary antibodies include against SPHK1 (1:1000, CST, 12071S, Danvers, MA, USA), PBX1 (1:1000, CST, 4342S, Danvers, MA, USA), Phospho-Akt (Ser 473; 1:1000, CST, 4060S, Danvers, MA, USA), Akt (1:1000, CST, 4685S, Danvers, MA, USA), Phospho-p44/42 MAPK (Thr202/Tyr204; 1:1000, CST, 4370S, Danvers, MA, USA), S1PR3 (1:1000, Abcam, ab126622, Cambridge, UK), S1PR1 (1:1000, Abcam, ab23386, Cambridge, UK), CDK4 (1:1000, Santa Cruz Biotechnology, sc-23896, Dallas, TX, USA), CDK2 (1:1000, Santa Cruz Biotechnology, sc-6248, Dallas, TX, USA), CDK1/CDK2 (1:1000, Santa Cruz Biotechnology, sc-53219, Dallas, TX, USA), CyclinD1 (1:1000, Santa Cruz Biotechnology, sc-8396, Dallas, TX, USA), β-actin (1:1000, Santa Cruz Biotechnology, sc-47778, Dallas, TX, USA), goat anti-rabbit and anti-mouse horseradish peroxidases (HRPs; 1:5000, Proteintech, B900610 and B900620, Chicago, IL, USA).
Ty 52156
Manufactured by Cayman Chemical
Sourced in United States
TY 52156 is a laboratory equipment product offered by Cayman Chemical. It is a device designed for use in scientific research and analysis, but a detailed description of its core function cannot be provided while maintaining an unbiased and factual approach. Further information may be available from the manufacturer.
Automatically generated - may contain errors
Lab products found in correlation
Jte 013, by Cayman Chemical (3 mentions)
Wortmannin, by Cayman Chemical (1 mentions)
Ab126622, by Abcam (1 mentions)
Sphingosine 1 phosphate (s1p), by Cayman Chemical (1 mentions)
Pd98059, by MedChemExpress (1 mentions)
Cdk1 cdk2, by Santa Cruz Biotechnology (1 mentions)
B900610, by Proteintech (1 mentions)
B900620, by Proteintech (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Antimycotic, by Thermo Fisher Scientific (1 mentions)
Wortmannin, by Thermo Fisher Scientific (1 mentions)
Easysep mouse neutrophil enrichment kit, by STEMCELL (1 mentions)
Baf a1, by Cell Signaling Technology (1 mentions)
L5293, by Merck Group (1 mentions)
Sew2871, by Cayman Chemical (1 mentions)
Amg9810, by Cayman Chemical (1 mentions)
Ex 26, by Bio-Techne (1 mentions)
Hc 030031, by Cayman Chemical (1 mentions)
Cym 5520, by Cayman Chemical (1 mentions)
5 protocols using ty 52156
1
Sphingosine-1-Phosphate Signaling Pathway
2
Neutrophil Isolation and Stimulation Assay
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Bone marrow–derived neutrophils were isolated from femurs/tibias by using the EasySep Mouse Neutrophil Enrichment Kit (19762, Stem Cell Technologies). Freshly isolated neutrophils were cultured (1.5 × 106 cells/mL in RPMI supplemented with 2% FBS and Antimycotic; 15240062, Thermo Fisher) and stimulated with S1P (SML2709, Millipore Sigma) or LPS (L5293, Millipore Sigma). In some experiments, neutrophils were pretreated with S1PR2 antagonist (JTE-013, 10009458, Cayman Chemical), S1PR3 antagonist (TY 52156, 19119, Cayman Chemical), autophagy antagonists, wortmannin (12-325, Fisher Scientific) or Baf A1 (54645S, Cell Signaling Technology), or CathB inhibitor (CA-074, 4846, Bio-Techne Corp.).
3
Detailed Compound Preparation for In Vitro and In Vivo Experiments
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S1P (d18:1), HC-030031, AMG 9810, SEW 2871, CYM 5520, CYM 5541, JTE 013, and TY 52156 were purchased from Cayman Chemical (MI, USA). Ex 26, CYM 50260, and A 971432 were purchased from Tocris Bioscience (MN, USA). Allyl isothiocyanate (AITC) and capsaicin were purchased from MilliporeSigma (MA, USA). S1P stock (10 mM, in DMSO) was used for in vitro experiments and 10 mM S1P stock in H2O filtered by 20 µm nylon filter (VWR International, LCC., PA, USA) was used for in vivo experiments. HC-030031 and AMG 9810 were stocked in DMSO (27.6 mM and 29.0 mM, respectively). Stock solutions were prepared as described. SEW2871 was dissolved in DMSO (50 mM). CYM 5520 and CYM 50260 were dissolved in 10 mM in DMSO. CYM5541, JTE 013, TY 52156, Ex 26, and A 971432 were prepared in 100 mM in DMSO. AITC and capsaicin were dissolved in ethanol (100 mM and 1 mM, respectively). All stocks were stored at −20°C.
4
Cell Culture and Treatments Protocol
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LAM patient-derived 621-101 and 621-103 cells [14 (link), 15 (link)], Eker rat uterine leiomyoma-derived (ELT3) cells [16 (link)], TSC2−/−p53−/−, TSC2+/+p53−/− MEFs, and HEK293T cells were cultured in DMEM with 10% FBS, 1% penicillin/streptomycin. and HEK293T cells. All cultures were incubated at 37 °C in a humidified 5% CO2 atmosphere. Rapamycin (20 nM; ENZO), PF543 (2 μM; Cayman), TY52156 (2 μM; Cayman), W123 (10 μM; Cayman), Spingosine-1-phosphate (2 μM; Cayman), were used as indicated.
5
Human Breast Cancer Cell Line Culture
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Human breast cancer cell lines, including MCF-7, SK-BR-3, MDA-MB-231, and LM2-4, were bought from American Type Culture Collection (ATCC, USA). MCF-7 cells were cultured in phenol-red free IMEM medium (Gibco BRL, USA). MDA-MB-231 and its metastatic variant LM2-4 cells were cultured in phenol-red free RPMI-1640 medium (Gibco BRL, USA). SK-BR-3 cells were maintained in DMEM (Dulbecco’s Modified Eagle’s Medium) (Gibco BRL, USA). All medium contained 10% heat-inactivated FBS (fetal bovine serum) (Gibco BRL, USA) and supplement 100x penicillin-streptomycin solution (LEAGENE), at 37°C in a 5% CO2 atmosphere. Human mammary cell line MCF-10A was used as the normal control. When breast cells or breast cancer cells grew to 80% to 90% convergence, the cells were used for passage or subsequent experiments. 5-FU and doxorubicin were purchased from Sigma (Sigma-Aldrich, USA). S1P was purchased from Enzo Life Sciences (Farmingdale, USA). Inhibitor of SphK1 PF543 was obtained from ApexBio (ApexBio, USA). TY52156, CAY10444, and JTE013 were purchased from Cayman Chemicals. In addition, LPA was obtained from Avanti Polar Lipids. SphK1 antibody was obtained from Exalpha Biologicals Inc.; GAPDH antibody was purchased from Cell Signaling Technology (CA, USA). Notch1 intracellular domain (N1ICD) antibody was obtained from Millipore (Merck Millipore, Germany).
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