Torin 1

Manufactured by MedChemExpress

Sourced in United States

Torin 1 is a potent and selective ATP-competitive inhibitor of the mammalian target of rapamycin (mTOR) kinase. It inhibits both mTOR complexes, mTORC1 and mTORC2, with high potency.

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Lab products found in correlation

Rapamycin, by Merck Group (2 mentions) Chloroquine, by Merck Group (2 mentions) Skov3, by American Type Culture Collection (1 mentions) Ovcar3, by American Type Culture Collection (1 mentions) Caov3, by American Type Culture Collection (1 mentions) Dulbecco modified eagle medium (dmem), by American Type Culture Collection (1 mentions) Rpmi 1640 medium, by American Type Culture Collection (1 mentions) Hyclone, by GE Healthcare (1 mentions) Sw626, by American Type Culture Collection (1 mentions) Mccoy s 5a medium, by American Type Culture Collection (1 mentions) Sb203580, by MedChemExpress (1 mentions) Hoecsht 33342, by Thermo Fisher Scientific (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Gemcitabine, by Pfizer (1 mentions) Rapamycin, by Apexbio (1 mentions) Uk5099, by Cayman Chemical (1 mentions) Cpi 613, by Cayman Chemical (1 mentions) Silvestrol, by Abcam (1 mentions) A 1155463, by Cayman Chemical (1 mentions) Fluorescence plate reader, by Molecular Devices (1 mentions)

8 protocols using torin 1

Ovarian Cancer Cell Lines Protocol

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Ovarian cancer cell lines OVCAR-3, Caov-3, SW626, and SK-OV-3 cell lines were purchased from American Type Culture Collection (Manassas, VA, USA). A2780 cell line was purchased from ECACC (UK). OVCAR-3 cells were cultured in RPMI-1640 Medium (ATCC, #30-2001) with 0.01 mg/ml bovine insulin and 20% fetal bovine serum (FBS, HyClone; GE Healthcare Life Science, Logan, UT, USA). Caov-3 cells were fostered in Dulbecco’s Modified Eagle’s Medium (DMEM, ATCC, #30-2001) with 10% FBS. SW626 cells were incubated in Leibovitz’s L-15 Medium (ATCC, #30-2008) with 10% FBS. SK-OV-3 cells were trained in McCoy’s 5a Medium (ATCC, #30-2007) with 10% fetal bovine serum. A2780 cells were planted in RPMI-1640 Medium (ATCC, #30-2001) containing 10% FBS. 100 U/mL penicillin and 100 μg/mL streptomycin were added to all mediums. A2780, OVCAR-3, Caov-3, and SK-OV-3 cells were maintained in an incubator at 37°C with 5% CO₂. SW626 cells were kept in an incubator at 37°C. SRT2183 (#HY-19759), SB203580 (#HY-10256), and Torin 1 (#HY-13003) were obtained from Med Chem Express (USA), chloroquine (#C6628), and rapamycin (#V900930) were purchased from Sigma (USA).

Sun T., Hu Y., He W., Shang Y., Yang X., Gong L., Zhang X., Gong P, & Yang G. (2020). SRT2183 impairs ovarian cancer by facilitating autophagy. Aging (Albany NY), 12(23), 24208-24218.

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Quantifying Cell Sensitivity to Pharmacological Agents

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For drug sensitivity assays, cells in the fertile culture were seeded on 12-well dishes at 18,000 cells/cm² and treated with selected drugs 24hrs later. The arid group was cultured for 0 (acute arid) or 14 (chronic arid) days under arid conditions prior to drug treatment. Cells supplemented daily for 4-5 days with fresh media supplemented with various drugs including: Gemcitabine (Pfizer), PHA-767491 (Cayman chemical), Silvestrol (Biovision), CPI-613 (Cayman chemical), UK 5099 (Cayman chemical) or A-1155463 (Cayman chemical) or vehicle (0.2% DMSO). Tumor cells were fixed at day 0 or endpoint (day 4-5) with paraformaldehyde 4% stained with 3 μM Hoecsht 33342 (Thermo) for 30’ at 37C and quantified using a fluorescence plate reader (Molecular devices) to assess cell numbers. Cell density was normalized for day 0 of each group.
For pharmacological inhibition of biosynthesis under serum repletion, treatment initiated with switch to arid conditions (acute arid) and cells were treated for 72hrs before readouts. Drugs used in that study include: Abemacicilib (MedChem Express), Rapamycin (ApexBio), Torin1 (MedChem Express) and Cycloheximide (Sigma).

Sela Y., Li J., Maheswaran S., Norgard R., Yuan S., Hubbi M., Doepner M., Xu J.P., Ho E., Measaros C., Sheehan C., Croley G., Muir A., Blair I.A., Shalem O., Dang C.V, & Stanger B.Z. (2022). Bcl-xL enforces a slow-cycling state necessary for survival in the nutrient-deprived microenvironment of pancreatic cancer. Cancer research, 82(10), 1890-1908.

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Macrophage Polarization and Signaling Inhibition

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BMDMs were obtained and cultured as previously described.25 (link) Raw 264.7 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 2 mmol/L L-glutamine. BMDMs or Raw 264.7 cells were stimulated with LPS (50 ng/mL, Sigma, St. Louis, MO, USA) and IFNγ (20 ng/mL, PeproTech, Rocky Hill, USA) or IL-4 (20 ng/mL, PeproTech) for 24 hours to induce M1 or M2 polarized macrophages. γ-secretase inhibitor IX (GSI, 30 µM, Sigma), BAY11-7082 (5 µM, Selleck), IKK-16 (0.5 µM, MedChem Express) or Torin 1 (100 nM, MedChem Express) was added to the medium, with DMSO as a control. The THP1 cells (ATCC) were cultured at 2×10⁵ cells/mL in RPMI 1640 medium supplemented with 10% FBS and 2 mmol/L L-glutamine, differentiating into macrophages with 200 nM phorbol 12-myristate 13-acetate (PMA, Sigma) treatment for 2 days. The cells were transfected with the respective oligos using Lipofectamine 2000 reagent (Invitrogen, Waltham, MA, USA), according to the manufacture’s protocol. The sequences of siRNAs against mTOR, NF-κB inhibitor κB-Ras2 (also known as Nkiras2) and IRF4 were shown in online supplementary table S3.

Wang L., Hu Y.Y., Zhao J.L., Huang F., Liang S.Q., Dong L., Chen Y., Yu H.C., Bai J., Yang J.M., Fan J.Y., Feng L., Li S.Z., Han H, & Qin H.Y. (2020). Targeted delivery of miR-99b reprograms tumor-associated macrophage phenotype leading to tumor regression. Journal for Immunotherapy of Cancer, 8(2), e000517.

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Glioblastoma Cell Lines and Inhibitors

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Human glioblastoma cell lines (LN229, A172, U87, and HUVEC) were obtained from China Infrastructure of Cell Line Resource (National Science and Technology Infrastructure, NSTI). Chloroquine was purchased from Sigma-Aldrich (St. Louis, MO, USA). Torin 1, Bafilomycin A1, and NTZ were all purchased from MedChem Express (MCE, USA).

Wang X., Shen C., Liu Z., Peng F., Chen X., Yang G., Zhang D., Yin Z., Ma J., Zheng Z., Zhao B., Liu H., Wang L., Wu J., Han D., Wang K., Zhong C., Hou X., Zhao W., Shu M., Wang X, & Zhao S. (2018). Nitazoxanide, an antiprotozoal drug, inhibits late-stage autophagy and promotes ING1-induced cell cycle arrest in glioblastoma. Cell Death & Disease, 9(10), 1032.

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Fibroblasts from CMT2A Patient

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Primary fibroblasts from a young patient affected by CMT2A^MFN2 (c.650G>T/p.Cys217Phe) and a healthy control (individual with no histological or biochemical signs of mitochondrial disease), were obtained as reported in ^{8 (link)} after informed consent. Cells were grown in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; EuroClone, ECB7501LX10) supplemented with 10% (v/v) fetal bovine serum (FBS; EuroClone, ECS5000L), 1% (v/v) L-glutamine (E EuroClone, ECB3000D), 1% (v/v) penicillin/streptomycin (EuroClone, ECB3001D), 50μg/ml of uridine (Sigma-Aldrich, U3003), in a humidified incubator at 37°C and 5% CO2 avoiding confluence at any time. All experiments were performed on cells with similar passage numbers, ranging from 5 to 8, to avoid any artefact due to senescence. For the experiments, growing cells were plated on sterile plastic dishes or flasks and allowed to adhere for at least 24 h before use. Torin1 (MedChemExpress, USA) was used at 0.1, 0.25 and 0.5 μM for 72 h, and DMSO as a vehicle.

Zanfardino P., Amati A., Petracca E.A., Santorelli F.M, & Petruzzella V. (2022). Torin1 restores proliferation rate in Charcot-Marie-Tooth disease type 2A cells harbouring MFN2 (mitofusin 2) mutation. Acta Myologica, 41(4), 201-206.

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Regulation of Stress Response Pathways

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Sangivamycin (HY-118384), rapamycin (HY-10219) and torin-1 (HY-13003) were obtained from MedChemExpress (NJ, USA). The primary antibodies used for western blotting were as follows: rabbit anti-ATF3 (#33593), rabbit anti-ATF4 (#118115) and rabbit anti-TRIB3 (#43043) were obtained from Cell signaling (MA, USA); rabbit anti-DDIT4 (#10638-1-AP) was obtained from Proteintech; mouse anti-actin (#sc-47778) was obtained from Santa Cruz Biotechnology (TX, USA). The ATF3 antibody (#18665) used for co-immunoprecipitation was obtained from Cell signaling (MA, USA). The primary antibodies used for immunochemistry staining: ki67 (#M7240, Agilent Technologies, CA, USA), p27 (#M72031, Agilent Technologies, CA, USA) and ATF3 (#MA5-31360, Thermo Scientific, MA, USA).

Lu X., Zhong L., Lindell E., Veanes M., Guo J., Zhao M., Salehi M., Swartling F.J., Chen X., Sjöblom T, & Zhang X. (2023). Identification of ATF3 as a novel protective signature of quiescent colorectal tumor cells. Cell Death & Disease, 14(10), 676.

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Modulating Autophagy in FaDu Cells

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For autophagy modulation, FaDu cells were treated for 24 h with 5 nM bafilomycin A1 (Sigma-Aldrich, B1793), 50 µM of hydroxychloroquine sulphate (Sigma-Aldrich, H0915), 100 µM of CPD18 (Calbiochem), 50 nM of autophinib (Sigma, SML2632), 10 µM of EACC (MedChemExpress), 200 nM of rapamycin (Sigma-Aldrich, R0395), 3 nM of Torin-1 (MedChemExpress), or 30 nM of NVP-BEZ235 (MedChemExpress). To induce starvation, cells were cultured in DMEM F12 without glutamine and without FBS (Biosera). Modulation of autophagy did not reduce the viability of FaDu cells (the viability ranged 98.1 to 99.9% across treatments).

Hanelova K., Raudenska M., Kratochvilova M., Navratil J., Vicar T., Bugajova M., Gumulec J., Masarik M, & Balvan J. (2023). Autophagy modulators influence the content of important signalling molecules in PS-positive extracellular vesicles. Cell Communication and Signaling : CCS, 21, 120.

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Multiparametric Analysis of Cellular Responses

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All reagents were dissolved and stored in DMSO or water. Baf-A1, Rapamycin, Torin-1, zVADfmk was obtained from Med Chem Express. DQ-BSA-red, FluoZin-3 AM, LysoTracker Red DND-99, BAPTA-am, EDTA, PI, and H2DCFDA were from Life Technologies. Clozapine, FTY720, NS8593, TTM, DIP, Annexin V/7-AAD, CQ, 3-MA, TPEN, 1,10 Phenanthroline were purchased from Sigma. Trypan blue was purchased from VWR. Naltriben was purchased from Santa Cruz Biotechnology.
Statistical Analysis. Data are presented as the means ± standard errors of the mean (SEMs) from at least three independent experiments. Statistical signi cance of differences was evaluated using ANOVA followed by Tukey's test. P values < 0.05 were considered statistically signi cant.

Xing Y., Wei X., Wang M.M., Liu Y., Sui Z., Wang X., Zhang Y., Fei Y.H., Jiang Y., Lu C., Zhang P., Chen R., Liu N., Wu M., Ding L., Wang Y., Guo F., Cao J.L., Qi J, & Wang W. (2022). Stimulating TRPM7 suppresses cancer cell proliferation and metastasis by inhibiting autophagy. Cancer letters, 525.

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