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Tepp 46

Manufactured by Cayman Chemical
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TEPP-46 is a small molecule inhibitor that targets the enzyme acetylcholinesterase. It is a laboratory reagent used in research applications.

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Lab products found in correlation

Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions) Rapamycin, by Thermo Fisher Scientific (2 mentions) Nk cell isolation kit 2, by Miltenyi Biotec (2 mentions) Il 12, by Miltenyi Biotec (2 mentions) Dehydroepiandrosterone dhea, by Merck Group (2 mentions) Glutamine, by Thermo Fisher Scientific (2 mentions) Il 15, by Thermo Fisher Scientific (2 mentions) Interleukin 2 (il 2), by Miltenyi Biotec (2 mentions) Il 15, by Miltenyi Biotec (2 mentions) 2 hydroxypropyl β cyclodextrin, by Cayman Chemical (1 mentions) Balb c nude mice, by Charles River Laboratories (1 mentions) N acetylcysteine, by Merck Group (1 mentions) Sodium dichloroacetate, by Merck Group (1 mentions) Oligomycin, by Merck Group (1 mentions) Carbonyl cyanide 3 chlorophenylhydrazone, by Merck Group (1 mentions) Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions) Dmem glutamax, by Thermo Fisher Scientific (1 mentions) 2 deoxyglucose, by Merck Group (1 mentions) Tamoxifen, by Merck Group (1 mentions) Metformin, by Cayman Chemical (1 mentions)

11 protocols using tepp 46

1

Characterization of Pancreatic Cancer Cell Lines

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Nine different human pancreatic cancer cell lines were purchased from RIKEN BioResource Centre (RIKEN BRC, Tsukuba, Japan) and PerkinElmer (Caliper LifeSciences, Hopkinton, MA, USA). PANC-1, PK-1, PK-45H, KLM-1, BxPc-3, PK45P, and PK-59 were maintained in RPMI-1640 medium, while MIA PaCa-2 was maintained in DMEM and KP-4 in DMEM/F12 medium. All media were supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 2mM glutamine. Cells were maintained in a humidified atmosphere of 21% O2, 5% CO2 at 37 °C.
The PKM2 activator IV, TEPP-46, was obtained from Cayman Chemicals (Ann Arbor, MI, USA) and the lactate dehydrogenase A inhibitor, FX-11, was obtained from Toronto Research Chemicals Inc. (Toronto, ON, Canada).
Mohammad G.H., Vassileva V., Acedo P., Olde Damink S.W., Malago M., Dhar D.K, & Pereira S.P. (2019). Targeting Pyruvate Kinase M2 and Lactate Dehydrogenase A Is an Effective Combination Strategy for the Treatment of Pancreatic Cancer. Cancers, 11(9), 1372.
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2

Establishing Subcutaneous and Orthotopic Brain Tumor Models

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Example 7

In vivo Tumor Models: For subcutaneous tumor models, U87 tumor cells (5×106 cells; 100 μL PBS) were injected subcutaneously on the back of female BALB/c nude mice (aged 6-8 weeks; Charles River Laboratories) and grown to approximately 150 mm3. Tumor dimensions were measured periodically using a caliper (by the same experienced researcher), with tumor volumes calculated by the equation: volume=(π/6)×a×b×c, where a, b, and c represent three orthogonal axes of the tumor. For orthotopic brain tumor models, 2×105 U87 cells were implanted 0.5 mm anterior and 2 mm to the right of the bregma in the brains of 6-8 week-old nude mice, held in place using a stereotactic unit. The cells, suspended in 4 μL PBS, were injected at a depth of 3 mm over 5 min with an AS blunt-ended Hamilton syringe, which was subsequently held in place for a further 5 min. Animals were anaesthetized with an i.p. injection of 150 mg/kg ketamine, 15 mg/kg xylazine. Mice were subsequently imaged by MRI and PET 32-35 days post intracranial injection and GBM39 tumor-bearing mice were imaged between 44-50 days after implantation. For blocking studies, mice were imaged with [11C]DASA-23 at baseline and then 24 hours later with a second tail-vein injection of [11C]DASA-23, 1 hour after i.p. injection of TEPP-46 [50 mg/kg in 40% w/v (2-hydroxypropyl)-β-cyclodextrin in water; about 3 μmol; Cayman Chemical].

US10039844B2. Imaging tumor glycolysis by non-invasive measurement of pyruvate kinase M2 (2018-08-07). The Board of Trustees of the Leland Stanford Junior University [US]. Inventors: Timothy Witney [GB], Michelle L. James [US], Sanjiv S. Gambhir [US].
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3

Isolation and Activation of NK Cells

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Splenocytes were isolated and cultured in IL-15 (15 ng/ml; PeproTech) at 37°C for 6 days. On day 4, the cells were supplemented with IL-15 (15 ng/ml) and cultured for an additional 2 d. On day 6, cultured NK cells were magnetically purified (NK cell Isolation Kit II, Miltenyi) and stimulated for 18 hr with IL-2 (20 ng/ml; National Cancer Institute Preclinical Repository) and/or IL-12 (10 ng/ml; Miltenyi Biotec) cytokines. Low-dose IL-15 (6.66 ng/ml) was added as a survival factor to unstimulated cultures. Experiments were carried out in the presence or absence of TEPP-46 (Cayman Chemical or EMD Millipore), rapamycin (20 nM; Fisher), N-acetyl-cysteine (7.5 mM; EMD Millipore) or dehydroepiandrosterone (DHEA) (75 μM, Sigma Aldrich). Splenocytes were cultured in RPMI medium containing 10% FBS, 2 mM glutamine (Thermo Fisher), 50 μM 2-ME (Sigma-Aldrich), and 1% Penicillin/Streptomycin (Thermo Fisher).
Walls J.F., Subleski J.J., Palmieri E.M., Gonzalez-Cotto M., Gardiner C.M., McVicar D.W, & Finlay D.K. (2020). Metabolic but not transcriptional regulation by PKM2 is important for natural killer cell responses. eLife, 9, e59166.
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4

Cytokine-Induced NK Cell Activation

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Splenocytes were isolated and cultured in IL-15 (15 ng/ml; PeproTech) at 37°C for 6 d. On day 4, the cells were supplemented with IL-15 (15 ng/ml) and cultured for an additional 2 d. On day 6, cultured NK cells were magnetically purified (NK cell Isolation Kit II, Miltenyi) and stimulated for 18 h with IL-2 (20 ng/ml; National Cancer Institute Preclinical Repository) and/or IL-12 (10 ng/ml; Miltenyi Biotec) cytokines. Low-dose IL-15 (6.66 ng/ml) was added as a survival factor to unstimulated cultures.
Experiments were carried out in the presence or absence of TEPP-46 (Cayman Chemical or EMD Millipore), rapamycin (20 nM; Fisher) or dehydroepiandrosterone (DHEA) (75 µM, Sigma Aldrich). Splenocytes were cultured in RPMI medium containing 10% FBS, 2 mM glutamine (Thermo Fisher), 50 μM 2-ME (Sigma-Aldrich), and 1% Penicillin/Streptomycin (Thermo Fisher).
Walls J., Subleski J., Palmieri E.M., Cotto M.G., Gardiner C.M., McVicar D.W., & Finlay D.K. (2020). Metabolic but not transcriptional regulation by PKM2 is important for Natural Killer cell responses.
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5

Cell Culture Conditions for Cancer Cell Lines

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All cell lines used in this study (MDA MB 231, MDA MB 468, HCC 1443, T47-D, MCF7, LnCap, PC3, and DU145) were kept in a humidified cell culture incubator at 37 °C with 5% CO2. Cells were also culture in Dulbecco’s Modified Eagle Medium (DMEM)-GlutaMAX™ (Gibco, Germany), supplemented with 10% FCS (v/v) (Gibco, Germany), 1% penicillin/streptomycin (v/v) (Gibco, Germany). MDA MB 468, HCC 1443, and T47-D are kind gifts from Dr. Stefan Wiemann (DKFZ, Heidelberg). Indicated drugs were added to the standard culture medium for the indicated time points. PKM2 allosteric activators DASA-58 and TEPP-46 (Cayman Chemical-Biomol GmbH, Germany), dehydroepiandrosterone (DHEA), CBR-5884, AZD-3965, metformin, and sodium oxamate (Cayman Chemical-Biomol GmbH, Germany) 2-deoxyglucose (Fluka-Sigma-Aldrich, Germany) Rotenon, carbonyl cyanide 3-chlorophenylhydrazone (CCCP), tamoxifen, oligomycin, and sodium dichloroacetate (Sigma Aldrich, Germany).
Almouhanna F., Blagojevic B., Can S., Ghanem A, & Wölfl S. (2021). Pharmacological activation of pyruvate kinase M2 reprograms glycolysis leading to TXNIP depletion and AMPK activation in breast cancer cells. Cancer & Metabolism, 9, 5.
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6

Optimal Wound Healing Compound Evaluation in C3H Mice

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Eight-week-old male C3H mice were purchased (Orient Bio Co., Seongnam-si, Republic of Korea). All animal experiments and procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of the Yonsei Laboratory Animal Research Center (IACUC-202102-1214-01, IACUC-202104-1243-01, and IACUC-202202-1417-01). Mice were housed under an automated environmental managing system in micro-ventilated cages (Threeshine Inc., Seoul, Republic of Korea). Mice were maintained under temperature-controlled (24 °C), humidity-controlled (40–70%) and light-controlled (standard 12 h light/dark cycle) conditions and provided with food and water. The mice were raised with a chow diet and bedding (Central Lab Animal Inc., Seoul, Republic of Korea). Considering the animal use alternatives (3Rs), we performed preliminary experiments to determine the optimal concentration of TEPP-46 (Cayman Chemical, Ann Arbor, MI, USA) and KY19382 [23 (link)] on wound healing in mice. Then, we performed minimal animal experiments under optimal conditions.
Kim E., Hwang Y., Kim H., Kim G.U., Ryu Y.C., Yoon M, & Choi K.Y. (2023). Pyruvate Kinase M2 Accelerates Cutaneous Wound Healing via Glycolysis and Wnt/β-Catenin Signaling. Pharmaceutics, 15(8), 2028.
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7

TEPP-46 Treatment Effect on Zebrafish Liver

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TEPP-46 was purchased from Cayman Chemical, USA. 10 μM TEPP-46 was used to treat zebrafish larvae from 4 dpf for for 96 hours. In all experimental groups, the survival rates at the end of experiment were always above 90%. After 96 hours treatment, images were taken and liver sizes were measured based on 2D images by using ImageJ (1.49j) as previously described [28 (link), 29 (link)]. The average liver sizes were calculated and summarized by using Graphpad Prism 6. Proliferation assay was performed by immunohistochemical staining on cryosections with anti-PCNA (Anaspec) as the primary antibody as previously described [7 (link)]. Images were taken for stained sections, and the number of total liver cells and proliferating cells were counted by using ImageJ (1.49j).
Li Z., Zheng W., Li H., Li C, & Gong Z. (2015). Synergistic Induction of Potential Warburg Effect in Zebrafish Hepatocellular Carcinoma by Co-Transgenic Expression of Myc and xmrk Oncogenes. PLoS ONE, 10(7), e0132319.
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8

Genetic Engineering of Cell Lines

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HEK293T and HeLa cells were cultured in DMEM containing 10% NCS. HCT116+/+ and HCT116−/− were cultured in McCoy′5A containing 10% FBS. Cells were transfected with plasmids using polyethylenimine (PEI, linear, 25KDa). AKT1S1 S202A, S202E, S203A and S203E were cloned into PRK7-Flag, PKM2 and K270M was cloned into pcDNA3.1-Flag, pcDNA3.1-Myc and pcDNA3.1-HA, respectively.
Chloroquine diphosphate salt (c6628-25 g) was from Sigma-aldrich, TEPP46 was from Cayman. Antibodies were either home-made or commercially purchased.
He C.L., Bian Y.Y., Xue Y., Liu Z.X., Zhou K.Q., Yao C.F., Lin Y., Zou H.F., Luo F.X., Qu Y.Y., Zhao J.Y., Ye M.L., Zhao S.M, & Xu W. (2016). Pyruvate Kinase M2 Activates mTORC1 by Phosphorylating AKT1S1. Scientific Reports, 6, 21524.
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9

Autoradiography of [18F]DASA-23 in Mice

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We completed autoradiography per previously described methods (27 (link)). For blocking studies, we administered [18F]DASA-23 1 h after intraperitoneal injection of TEPP-46 (50 mg/kg in 40% w/v (2-hydroxypropyl)-b-cyclodextrin in PBS; Cayman Chemical, Ann Arbor, MI). Thirty min after injection of radioactivity, we transcardially perfused mice with saline (15 mL) and the brain embedded in optimal cutting temperature (OCT) and processed for autoradiography according to published methods (27 (link)).
Beinat C., Patel C.B., Haywood T., Murty S., Naya L., Castillo J.B., Reyes S.T., Phillips M., Buccino P., Shen B., Park J.H., Koran M.E., Alam I.S., James M.L., Holley D., Halbert K., Gandhi H., He J.Q., Granucci M., Johnson E., Liu D.D., Uchida N., Sinha R., Chu P., Born D.E., Warnock G.I., Weissman I., Hayden-Gephart M., Khalighi M., Massoud T.F., Iagaru A., Davidzon G., Thomas R., Nagpal S., Recht L.D, & Gambhir S.S. (2021). A Clinical PET Imaging Tracer ([18F]DASA-23) to Monitor Pyruvate Kinase M2 Induced Glycolytic Reprogramming in Glioblastoma. Clinical cancer research : an official journal of the American Association for Cancer Research, 27(23), 6467-6478.
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10

Isolation and Culture of Primary Mouse Tracheal Epithelial Cells

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Primary mouse tracheal epithelial cells (MTE) were isolated from WT C57BL/6NJ mice (purchased from The Jackson Laboratory, Bar Harbor, ME) and cultured as previously described (15 (link), 16 (link)). WT MTE cells were grown on 12-well transwell inserts to confluency, followed by overnight starvation, and pre-treated with 100 μM TEPP46 (Cayman Chemicals) for 1 hour prior to stimulation with 10 ng/mL IL-1β (R&D Systems) for 24 hrs. In all cell experiments, MTE cells were treated at the apical and basolateral side. In HDM-treated MTE cell experiments, cells were washed 24 hours after IL-1β treatment, incubated with DMEM/F12 medium for 2 hours before stimulation with 50 μM HDM (GREER, Lenoir, NC, XPB70D3A2.5, lot 348718; volume: 2.5 mL/vial; endotoxin: 1140 EU/vial, Der p 1 levels: 144.9 mcg/vial; dry weight: 17 mg/vial; protein 2.92 mg/vial) for an additional 2 hours. Cells were harvested for protein or mRNA, and medium was collected for analysis of lactate and cytokine levels. To examine the contribution of phosphorylation of STAT3, MTE cells were treated with Stattic (17 (link)) (Abcam, Cambridge, UK) at the indicated concentrations for 1 hour prior to IL-1β treatment for 24 hrs.
van de Wetering C., Aboushousha R., Manuel A.M., Chia S.B., Erickson C., MacPherson M.B., van der Velden J.L., Anathy V., Dixon A.E., Irvin C.G., Poynter M.E., van der Vliet A., Wouters E.F., Reynaert N.L, & Janssen-Heininger Y.M. (2020). Pyruvate Kinase M2 Promotes Expression of Proinflammatory Mediators in House Dust Mite–Induced Allergic Airways Disease. The Journal of Immunology Author Choice, 204(4), 763-774.
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