NOD/SCID and 69 athymic nude mice were purchased from The Jackson Laboratory and from Envigo Rms, Inc., respectively. All drugs (except LY3214996, and Navire 13909) were received via CTEP agreement (U54 NCI). LY3214996 was purchased from MedChem Express. Navire 13909 (SHP2 inhibitor) was kindly provided by Navire Pharma under an MTA. All drugs (except palbociclib) were dissolved in dimethyl sulfoxide (DMSO) and stored at −20°C. For in vivo experiments, all drugs were prepared fresh daily from DMSO stocks or powder (for palbociclib). List of antibodies and other reagents used in this study are provided in Supplementary Table S1.
Ly3214996
Manufactured by MedChemExpress
Sourced in United States
LY3214996 is a chemical compound used as a laboratory reagent. It functions as a selective inhibitor of the enzyme glutaminase. The core purpose of LY3214996 is to serve as a research tool for studying the role of glutaminase in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Nod scid, by Jackson ImmunoResearch (1 mentions)
Collagen 1, by BD (1 mentions)
Laminin, by BD (1 mentions)
Plasmin, by Abcam (1 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Sartorius (1 mentions)
Ampk sirna, by Santa Cruz Biotechnology (1 mentions)
Oxaliplatin o9512, by Merck Group (1 mentions)
Antibiotic antimycotic, by Thermo Fisher Scientific (1 mentions)
Ax 15836, by MedChemExpress (1 mentions)
Lps escherichia coli o55 b5, by MedChemExpress (1 mentions)
P erk5, by Cell Signaling Technology (1 mentions)
Tnf α, by Cusabio (1 mentions)
Cxcl8, by Thermo Fisher Scientific (1 mentions)
Bay 11 7082, by MedChemExpress (1 mentions)
Stattic, by MedChemExpress (1 mentions)
Recombinant human lif, by Thermo Fisher Scientific (1 mentions)
8 protocols using ly3214996
1
Xenograft Mouse Models for Drug Evaluation
2
Satellite Cell Proliferation Assay
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Satellite cells were isolated as described above and seeded in plates coated with collagen I (1 μg mL−1, BD Bioscience) and laminin (10 μg mL−1, BD Bioscience) in myogenic growth media (20% heat-inactivated horse serum, 1% pencillin/streptomycin and 5 ng mL−1 bFGF in F10). 384-well plates were used for all EdU proliferation assays using ~2,000 cells per well. 24-well plates were used for signaling experiments using ~150,000 cells. Cells were treated with recombinant plasminogen (Abcam) at 5 μg/ml or 54 nmol/L or plasmin (Abcam) at 5 μg/ml or 57 nmol/L. Additional groups were treated with the ERK inhibitor LY3214996 (MedChemExpress) at 2.3 μg/ml or 5 μmol/L.
3
Investigating Colorectal Cancer Cell Lines
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The human CRC cell lines, HCT116 and LoVo, were purchased from the Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, which were cultured in Dulbecco's modified Eagle medium (Gibco TM, 11965092) with 10% fetal bovine serum (Biological Industries, 04‐001‐1A‐US), 100 U/mL penicillin, and 100 mg/mL streptomycin (Gibco TM, 15140‐122) at 37°C in an atmosphere of 5% CO2. Oxaliplatin (O9512), metformin (PHR1084), insulin (1342106), AMPK inhibitor, and compound C (171260) were purchased from Sigma‐Aldrich. LY3214996 was purchased from MedChem Express. AMPK siRNA was purchased from Santa Cruz Biotechnology. Cell Counting Kit‐8 (KGA317s‐500) was purchased from KeyGen. For transfection, HCT116 cells were seeded in 6‐well plates at 70% confluency, and then transfected with AMPK siRNA (5 nmol/L) using Lipofectamine 2000 (Invitrogen/Thermo‐Fisher Scientific) according to the manufacturer's instructions. The knockdown efficiency at the optimized concentration was < 1%.
4
Punicalagin Modulates Inflammatory Signaling
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LPS (Escherichia coli O55:B5), LY3214996, AX15836, and p38 IN-1 were purchased from MedChemExpress LLC (Shanghai, China). Punicalagin [>98% high-performance liquid chromatography (HPLC) purity] was purchased from Tauto Biotech (Shanghai, China). Fetal bovine serum (FBS), Dulbecco's modified Eagle medium (DMEM), and antibiotic-antimycotic were obtained from Gibco (Grand Island, NY, USA). The ELISA kits for IL-1β and TNF-α were bought from Cusabio (Wuhan, China). Bicinchoninic acid (BCA) protein assay kit was purchased from Pierce (Rockford, IL, USA). Antibodies for GAPDH, PTP1B, MKP1, p-ERK1/2, p-ERK5, p-p38, iNOS, and Arg-1 were produced by Cell Signaling Technology (Danvers, MA, USA). The goat anti-mouse antibody was purchased from Li-cdr Odyssye® (Lincoln, NE, USA). The assay kit used was manufactured by RayBiotech, Inc. (Norcross, GA, USA).
5
Modulation of Cell Signaling Pathways
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Recombinant human LIF (#300-05, 20 ng/mL), CXCL3 (#300-40, 20 ng/mL) and CXCL8 (#200-08M, 20 ng/mL) were all purchased from Peprotech. LIF neutralizing antibody (AB-250-NA, 2 μg/mL) was purchased from R&D, and CXCL3 neutralizing antibody (PP1014P2, 5 μg/mL) was purchased from Origene. Stattic (#HY-13818), LY3214996 (#HY-101494), PD98059 (#HY-12028), SB 203580 (#HY-10256), LY294002 (#HY-10108), JSH-23 (#HY-13982), BAY 11-7082 (#HY-13453), T-5224 (#HY-12270), TK216 (#HY-122903), TAT-DEF-Elk-1 (#HY-P2262A), SB225002 (#HY-16711), Reparixin (#HY-15251), and EC330 (#HY-100949) were all purchased from Med Chem Express. SB-505124 (#M2250) was purchased from Abmole. A list of the concentrations and targets used for the inhibitors is provided in Supplementary Table 1 .
6
Signaling pathway modulation assays
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The cells were transfected with specific siRNAs or plasmids using Lipofectamine 2000 (Invitrogen, CA, USA) according to the manufacturer's protocol. For EGF treatment, the cells were starved in medium containing 0.1% serum for 24 h, and then, 100 ng/ml EGF was added to the culture. In some cases, the specific inhibitors AG1478, salirasib, TAK580, LY3009120 and LY3214996 from MedChem Express (NJ, USA) and U0126 and LY294002 from Cell Signaling Technology (CST, MA, USA) were used to treat the cells for the indicated times. BC-1471 was purchased from AOBIOUS (Cat No: AOB36470, MA, USA).
7
Chemical Compounds Acquisition and Handling
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Simmiparib was synthesized at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences. Olaparib, G007‐LK, VE‐821, chloroquine, losmapimod, RBN‐2397, cisplatin, and LY3214996 were purchased from MedChemExpress (NJ, USA). Talazoparib, veliparib, irinotecan, and rucaparib were purchased from Selleck Chemicals (Shanghai, China). All drugs were dissolved in DMSO and stored at −20°C.
8
Signaling Pathway Inhibitors and Autophagy Markers
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LPS (#L2880), SB230580 (#S8307), Anisomycin (#A5862), SP600125 (#S5567), and MG132 (#474790) were purchased from Sigma-Aldrich. LY3214996 (#HY100494), Bafilomycin A1 (#HY-100558), and Cycloheximide (#HY-12320) were purchased from MedChem Express. The following primary antibodies were used: anti-p38 MAPK (#9217, Cell Signaling), antiphospho-p38 MAPK (#9216, Cell Signaling), anti-NLRP3 (#15101, Cell Signaling), anti-IL1 (#12703, Cell Signaling), anti-Atg13 (#13273, Cell Signaling), anti-LC3 (#8899, Cell Signaling), anti-p62 (#5114 Cell Signaling), anti-ULK1 (#8054, Cell Signaling), anti-FIP200 (#12436, Cell Signaling), anti-ubiquitin (#134953, Abcam), anti-Atg5 (#9980, Cell Signaling), anti-Myc (#2276, Cell Signaling), anti-GST (#2625, Cell Signaling), and β-actin as a loading control (#4970, Cell Signaling).
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