Compound 968

Manufactured by Merck Group

Sourced in United Kingdom

Compound 968 is a chemical compound developed by Merck Group for use in scientific research and laboratory settings. It is a small molecule that serves as a tool compound for in vitro and in vivo studies. The core function of Compound 968 is to act as a research reagent, without further interpretation or extrapolation on its intended use.

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Lab products found in correlation

Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions) Rapamycin, by Merck Group (1 mentions) 6 aminonicotinamide, by Merck Group (1 mentions) Lc ms grade water, by Thermo Fisher Scientific (1 mentions) High glucose dmem, by Thermo Fisher Scientific (1 mentions) Hplc grade isopropanol, by Thermo Fisher Scientific (1 mentions) X bridge amide hilic column, by Waters Corporation (1 mentions) Methyl tertiary butyl ether, by Merck Group (1 mentions) 2 deoxyglucose, by Merck Group (1 mentions) Lc ms grade acetonitrile, by Thermo Fisher Scientific (1 mentions) Cadenza cd c18 column, by Imtakt (1 mentions) Cb 839, by Selleck Chemicals (1 mentions) Alkbh5, by Abcam (1 mentions) Ythdf2, by Proteintech (1 mentions) Ddit4, by Proteintech (1 mentions) P p70s6k, by Cell Signaling Technology (1 mentions) Mettl14, by ABclonal (1 mentions) Actinomycin d, by Selleck Chemicals (1 mentions) Chloroquine cq, by Merck Group (1 mentions) Mettl3, by ABclonal (1 mentions)

7 protocols using compound 968

Metabolite Extraction and Quantification Workflow

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Methyl tertiary-butyl ether (MTBE) used for lipid and metabolite extraction was purchased from Sigma-Aldrich. LC-MS grade water, LC-MS grade acetonitrile (ACN), and HPLC grade isopropanol (IPA) were purchased from Fisher Chemical. HPLC grade methanol (MeOH) was purchased from Pharmco-Aaper. Mobile phase buffer LC-MS grade formic acid and formic acetate were purchased from Sigma-Aldrich. Fully carbon labeled ¹³C₆ glucose for isotopic labeling was purchased from Cambridge Isotope Laboratories. High glucose DMEM and glucose-free Dulbecco’s modified eagle medium (DMEM) were purchased from Gibco. For the drug treatments, 6-aminonicotinamide, 2-deoxyglucose, compound 968 and rapamycin were purchased from Sigma-Aldrich. A Cadenza CD-C₁₈ column (150 × 2 mm) was purchased from Imtakt, and an XBridge Amide HILIC column (3.5 µm, 4.6 × 100 mm) was purchased from Waters.

Huang H., Yuan M., Seitzer P., Ludwigsen S, & Asara J.M. (2020). IsoSearch: An Untargeted and Unbiased Metabolite and Lipid Isotopomer Tracing Strategy from HR-LC-MS/MS Datasets. Methods and Protocols, 3(3), 54.

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Molecular Mechanisms of mRNA Modification

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ATF4 (rabbit, 11815S), p-mTOR (rabbit, 5536S), mTOR (rabbit, 2983S), p-p70S6K (rabbit, 9205S), p70S6K (rabbit, 2708S), pS6 (rabbit, 4857S), LC3B (rabbit, 3868S), and sequestosome 1 (SQSTM1, rabbit, 8025) monoclonal antibodies were purchased from Cell Signaling Technology (Shanghai, China). DDIT4 (rabbit, 10638-1-AP) and YTHDF2 (rabbit, 24744-1-AP) polyclonal antibodies were purchased from Proteintech. FTO (rabbit, ab126605), WTAP (rabbit, ab195380), ALKBH5 (rabbit, ab69325), and m⁶A (mouse, ab208577) antibodies were purchased from Abcam. METTL3 (rabbit, a8370), and METTL14 (rabbit, a8530) polyclonal antibodies were purchased from ABclonal. Compound 968 was purchased from Sigma-Aldrich (SML1327). Actinomycin D was purchased from Selleck Chemicals (S8964). Chloroquine (CQ) was obtained from Sigma-Aldrich (C6628). Meclofenamate sodium was purchased from MCE (HY-B1320). CB839 was purchased from Selleck Chemicals (S7655).

Han S., Zhu L., Zhu Y., Meng Y., Li J., Song P., Yousafzai N.A., Feng L., Chen M., Wang Y., Jin H, & Wang X. (2021). Targeting ATF4-dependent pro-survival autophagy to synergize glutaminolysis inhibition. Theranostics, 11(17), 8464-8479.

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Establishment and Characterization of Breast Cancer Cell Lines

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LCC1 (sensitive), LCC2 (TAM resistant; ICI sensitive), and LCC9 (ICI resistant and TAM cross-resistant) and LY2 (LY 117018 [Raloxifene analog] resistant and TAM and ICI cross-resistant) cells were established as previously described [17 (link), 18 (link)]. Cells were grown in phenol red–free IMEM (Life Technologies, Grand Island, NY; A10488-01) with 5% charcoal-stripped calf serum (CCS); this media contains 2 mM L-glutamine and ~12 mM glucose. For glucose/glutamine-dependency assays, DMEM without glucose or glutamine (Life Technologies; A14430-01) was used supplemented with 5% CCS. LCC9Gln were derived from LCC9: cells were grown in DMEM without glucose but containing 2 mM L-glutamine (glutamine-only media) for 72 h; cells that survived (<5%) were continually grown in glutamine-only media for 12 weeks. All cells were authenticated by DNA fingerprinting and tested regularly for Mycoplasma infection. Faslodex and STF-31 were obtained from Tocris Bioscience (Ellisville, MO). Compound-968 was purchased from EMD Millipore (Billerica, MA). 10058-F4 was kindly provided by Dr. Steven Metallo (Georgetown University, Department of Chemistry). All other chemicals were purchased from Sigma-Aldrich.

Shajahan-Haq A.N., Cook K.L., Schwartz-Roberts J.L., Eltayeb A.E., Demas D.M., Warri A.M., Facey C.O., Hilakivi-Clarke L.A, & Clarke R. (2014). MYC regulates the unfolded protein response and glucose and glutamine uptake in endocrine resistant breast cancer. Molecular Cancer, 13, 239.

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Colorectal Cancer Cell Line Culture

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Human colorectal cancer cell lines were purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 (Invitrogen, 11875-093) or DMEM (Invitrogen, 11965-092) medium supplemented with 10% fetal bovine serum (FBS). All cells were incubated at 37°C with 5% CO2 and 95% humidity. The following antibodies were used: cleaved caspase 3 (9661-s, CST), cleaved PARP (9541-s, CST), LC3B (Nb100-2220, NOVUS), SQSTM1 (pm045, MBL), β-actin (4970L, CST). Compound 968 was purchased from Merck Millipore (352010). Chloroquine (C6628) was obtained from Sigma while L-ASP was from Slpharm.

Li J., Song P., Zhu L., Aziz N., Zhou Q., Zhang Y., Xu W., Feng L., Chen D., Wang X, & Jin H. (2017). Synthetic lethality of glutaminolysis inhibition, autophagy inactivation and asparagine depletion in colon cancer. Oncotarget, 8(26), 42664-42672.

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Preparing Reagents for Cell Assays

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Cisplatin (Wako), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich), and Compound 968 (Merck Millipore) were dissolved in phosphate-buffered saline (PBS) and filtered through a 0.22-μm filter. 5-aza-2′-deoxycytidine (5-aza-dC; Tokyo chemical industry) was initially dissolved in dimethyl sulfoxide (DMSO) and further diluted with culture medium.

Guo J., Satoh K., Tabata S., Mori M., Tomita M, & Soga T. (2021). Reprogramming of glutamine metabolism via glutamine synthetase silencing induces cisplatin resistance in A2780 ovarian cancer cells. BMC Cancer, 21, 174.

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Glutaminase Inhibitor for Extracellular Vesicle Harvesting

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A glutaminase inhibitor, compound 968 (968; EMD Millipore, Billerica, MA), was prepared by dissolving 968 in dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO) at 30 mM. 36 hours prior to ESV harvesting, cells were treated with 968 at 10 μM (71 (link), 72 (link)); a total of 1.67 μL of 968 in DMSO was added to 5 mL of culture medium (0.03% by volume). Following the protocol described in Section 2.3, cells were treated with 10 μM 968 under serum-starved conditions for 12 hours. Subsequent harvesting and centrifugation steps are identical to those previously described.

Santana S.M., Antonyak M.A., Cerione R.A, & Kirby B.J. (2014). Cancerous Epithelial Cell Lines Shed Extracellular Vesicles With a Bimodal Size Distribution that is Sensitive to Glutamine Inhibition. Physical biology, 11(6), 065001.

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Dissolution of Metabolic Inhibitors

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BPTES, UK-5099 (both Sigma-Aldrich, Inc.) and Compound 968 (Merck Inc.) were dissolved in DMSO. Etomoxir (Sigma-Aldrich, Inc.) was prepared as a water solution.

Rupprecht A., Moldzio R., Mödl B, & Pohl E.E. (2019). Glutamine regulates mitochondrial uncoupling protein 2 to promote glutaminolysis in neuroblastoma cells. Biochimica et biophysica acta. Bioenergetics, 1860(5), 391-401.

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