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Mln0128

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MLN0128 is a small molecule inhibitor that targets the mTOR (mechanistic target of rapamycin) pathway. It functions as a potent and selective inhibitor of the mTOR kinase, which plays a central role in regulating cell growth, proliferation, and metabolism.

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Lab products found in correlation

Rapamycin, by Selleck Chemicals (3 mentions) Mk 2206, by Selleck Chemicals (2 mentions) β actin, by Cell Signaling Technology (2 mentions) Fetal bovine serum (fbs), by Merck Group (2 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions) Wortmannin, by Merck Group (1 mentions) U 73122 hydrate, by Merck Group (1 mentions) Sb202190, by Santa Cruz Biotechnology (1 mentions) Bapta am, by Focus Biomolecules (1 mentions) Gw5074, by Santa Cruz Biotechnology (1 mentions) Bay11 7082, by InvivoGen (1 mentions) Bafilomycin a, by Merck Group (1 mentions) Celastrol, by InvivoGen (1 mentions) Sb203580, by Selleck Chemicals (1 mentions) Fk506, by Merck Group (1 mentions) Nvp bez235, by Selleck Chemicals (1 mentions) Ku 0063794, by Merck Group (1 mentions) Rapamycin, by Cell Signaling Technology (1 mentions) Effectene transfection reagent, by Qiagen (1 mentions) Insulin, by Eli Lilly (1 mentions)

9 protocols using mln0128

1

Signaling Pathway Inhibitors in Neutrophils

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To study the underlying signalling NET pathway, several chemical inhibitors were used. Before stimulation, neutrophils were pre-treated for 30 min at 37 °C in RPMI 10% with DMSO (vehicle control), 1 µM Wortmannin (PI3K inhibitor; Sigma-Aldrich), 1 µM R406 (SYK inhibitor; Selleckchem), 10 µM SB203580 (p38 MAPK inhibitor; Selleckchem), 10 µM SB202190 (p38 MAPK inhibitor; Santa Cruz Biotechnology) 10 µM MLN0128 (pan-mTORC inhibitor; Selleckchem), 10 µM BAPTA-AM (chelator of Ca2+;Focus Biomolecules), 1 µM DPI (NADPH oxidase inhibitor; Sigma-Aldrich), 1 µM Rapamycin (mTORC1 inhibitor; Selleckchem), 1 µM Celastrol (NF-κB inhibitor; InvivoGen), 0.1 µM Bay-11-7082 (IκB phosphorylation inhibitor; InvivoGen), 0.1 µM GW5074 (cRaf1 kinase inhibitor; Santa Cruz Biotechnology), 0.1 µM U-73122 hydrate (PLC inhibitor; Sigma-Aldrich), 1 nM Bafilomycin A (vacuolar-type H+ -ATPase inhibitor; Sigma-Aldrich), 0.1 µM JNKi II 420128 (JNK inhibitor; Merck Millipore) or 0.1 µM Mytoquinone (mitochondrial ROS inhibitor; BIOTREND).
van der Linden M., Westerlaken G.H., van der Vlist M., van Montfrans J, & Meyaard L. (2017). Differential Signalling and Kinetics of Neutrophil Extracellular Trap Release Revealed by Quantitative Live Imaging. Scientific Reports, 7, 6529.
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2

Glioma Cell Line Transfection and Transduction

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Human glioma cell lines were grown in 10% FBS. These included LN229, U87MG, GBM43, GBM5, and GBM12 (Sarkaria et al., 2006 (link)). Plasmids pcDNA3-mTORWT, pcDNA3-mTORR2505P, pcDNA3-mTORS2215Y (Sato et al., 2010 (link)) were obtained from Addgene (plasmids #26036-8); and transfected stably into LN229 cells using Effectene-transfection reagent (Qiagen). To generate retrovirus to transduce LN229 and U87MG with EGFR or EGFRvIII (Fan et al., 2007 (link)), the packaging cell line 293T was co-transfected with pWLZ-hygro-EGFR plasmid gag/pol and VSVg or with pLRNL-neo-EGFRvIII plasmid gag/pol and VSVg again using Effectene. High-titer virus was collected at 48 hr and used to infect cells as described (Fan et al., 2006 (link)). Transfected and transduced cells were selected as pools with G418 (800 μg/ml) or hygromycin (500 μg/ml) for two weeks. Inhibitors INK1437, TGX221, IC87114, INK1358, AS252424, AS605240, and GDC-0941 were from Pingda Ren and Liansheng Li. AKT inhibitor VIII was from EMD Biosciences. Rapamycin was from Cell Signaling. NVP-BEZ235, MLN0128, and MK-2206 were from Selleck Chemicals. KU-0063794 and FK-506 were from Sigma-Aldrich Inc. Insulin was from Eli Lilly & Co. D-luciferin was from Gold Biotechnology USA. RapaLink-1 and RapaLink-2 were synthesized by CM, MO, and KMS as described (Rodrik-Outmezguine et al., 2016 (link)).
Fan Q., Aksoy O., Wong R.A., Ilkhanizadeh S., Novotny C.J., Gustafson W.C., Truong A.Y., Cavanan G., Simonds E.F., Haas-Kogan D., Phillips J.J., Nicolaides T., Okaniwa M., Shokat K.M, & Weiss W.A. (2017). A Kinase Inhibitor Targeted to mTORC1 Drives Regression in Glioblastoma. Cancer cell, 31(3), 424-435.
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3

Phosphorylation Signaling Pathway Analysis

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Anti-mouse S6K, S6, 4EBP, PDCD4, beta-Actin, phospho-S6K, phosphor-S6, phosphor-4EBP, and phospho-4EBP-Alexa647 antibodies were purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-mouse phoshoS6-PE, anti-mouse CD45.1 Pacific Blue, and anti-mouse Ki67 Brilliant Violet 605 antibodies were purchased from Biolegend (San Diego, CA, USA). Anti-GFP antibody purchased from Abcam (Cambridge, UK). Cytarabine arabinoside (Ara-C) and doxorubicin were purchased form Sigma-Aldrich (St. Louis, MO, USA). Rapamycin, Rad001, Torin2, AZD0855, and MLN(0128) were purchased from Selleckchem (Houston, TX, USA).
Oki T., Mercier F., Kato H., Jung Y., McDonald T.O., Spencer J.A., Mazzola M.C., van Gastel N., Lin C.P., Michor F., Kitamura T, & Scadden D.T. (2021). Imaging dynamic mTORC1 pathway activity in vivo reveals marked shifts that support time-specific inhibitor therapy in AML. Nature Communications, 12, 245.
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4

Evaluation of Rapamycin and MLN0128 Compounds

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MLN0128 and rapamycin were purchased from Selleckchem. Compounds were dissolved in DMSO (Sigma-Aldrich), aliquoted, and stored at −80°C. Cell lines were cultured in RPMI 1640 supplemented with 100 U/ml penicillin-streptomycin (Thermo Fisher), 2 mM l-glutamine (Thermo Fisher), and 10% fetal bovine serum (FBS) (Sigma-Aldrich), maintained at 37°C in 5% CO2, and passaged for no more than 3 months. BC-1, BCBL-1, BC-3, and BCP-1 were obtained from ATCC. BCBL-1TrexRTA cells were a gift from J. Jung (46 (link)). RedFect (Perkin Elmer) was used to confer continuous red-luciferase expression (BCBL-1TrexRTA-luc), and cells were maintained in hygromycin B (20 µg/ml) and puromycin (1.25 µg/ml). Cell lines were authenticated by NextGen-based HLA and short tandem repeat (STR) typing (https://www.med.unc.edu/vironomics) and found free of mycoplasma by the Mycoalert mycoplasma test kit (Lonza).
Caro-Vegas C., Bailey A., Bigi R., Damania B, & Dittmer D.P. (2019). Targeting mTOR with MLN0128 Overcomes Rapamycin and Chemoresistant Primary Effusion Lymphoma. mBio, 10(1), e02871-18.
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5

In Vitro and In Vivo Compound Preparation

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For in vitro studies MLN0128 (Selleckchem Houston, TX, USA) was dissolved in DMSO, phenfomin was dissolved in DMEM. For in vivo studies MLN0128 was dissolved in 1-methyl-2-pyrrolidinone (NMP), then diluted in 15% PEG diluted in water; phenformin was diluted in water at 1.8 mg/ml. Rapamycin was purchased from LC Laboratories (Woburn, MA) and dissolved in DMSO. Antibodies from Cell Signaling Technology (Beverly, MA, USA) used for immunoblots were diluted 1:1,000 and included phospho-p70 S6 kinase (Thr389 #9234), phospho-S6 (Ser235/236 #4858), total 4E-BP1 (#9644), phospho-4E-BP1 (Thr37/46 #2855), phospho-Akt (Ser473 #4060), phospho-Akt (Thr308 #4056), phospho-NDRG1 (Thr346 #5482), Phospho-Tuberin/TSC2 (Thr1462 #3611), phospho-GSKα/β (Ser21/9 #9331), beta-actin (#4970), cleaved PARP (Asp214 #5625), cleaved caspase 3 (Asp175 #9661) and phospho-Raptor (Ser792 #2083). Anti-Hif-1alpha (C-Term #10006421 1:200) antibody was purchased from Cayman Chemical and anti-GLUT1 (GT11-A 1:1,000) antibody was purchased from Alpha Diagnostic International (San Antonio, TX, USA). Plasmid expressing dox-inducible 4EBP1 4Ala (pCW57.1-4EBP1_4xAla, plasmid # 38240) and control vector (pCW57.1, plasmid # 41393) were purchased from Addgene.
Momcilovic M., McMickle R., Abt E., Seki A., Simko S.A., Magyar C., Stout D.B., Fishbein M.C., Walser T.C., Dubinett S.M, & Shackelford D.B. (2015). Heightening energetic stress selectively targets LKB1-deficient non-small cell lung cancers. Cancer research, 75(22), 4910-4922.
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6

EMT Induction in Mammary Epithelial Cells

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NMuMG cells were cultured in DMEM with 10% FBS (Sigma-Aldrich Co. LLC.), 10 μg/ml insulin (Sigma-Aldrich Co. LLC), 100 U/ml penicillin, and 100 μg/ml streptomycin. Immortalized human mammary epithelial (HMLE) cells were from Jing Yang (UCSD) and maintained in MGEM (Lonza), as described (20 (link)). To induce EMT, NMuMG cells were treated with 1 ng/ml TGF-β (R&D), and HMLE cells were treated with 2 ng/ml TGF-β (R&D), as indicated. SB431542 was from Sigma-Aldrich and was used at 5 μM. MK2206 and MLN0128 were from Selleck Chemicals and were used at 500 nM and 100 nM respectively. RapaLink-1 was synthesized as described (32 (link)), and was used in cell culture at 5 nM. Doxorubicin and 4-hydroperoxycyclophosphamide was from Toronto Research Chemicals. Cisplatin and 5-fluorouracil were from Wako. Paclitaxel was from Bristol-Myers.
Katsuno Y., Meyer D.S., Zhang Z., Shokat K.M., Akhurst R.J., Miyazono K, & Derynck R. (2019). Chronic TGF-β exposure drives stabilized EMT, tumor stemness, and cancer drug resistance with vulnerability to bitopic mTOR inhibition. Science signaling, 12(570), eaau8544.
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7

Antibodies and Reagents for SLFN11 Study

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The following antibodies were used in our study: anti-SLFN11 (Atlas antibodies, HPA023030), anti-GAPDH (Sigma-Aldrich), anti-RPS4X (Abcam), anti-phospho-RPS6 or anti-phospho-S6 (Ser235/236; Cell Signaling Technology), anti-RPS6 (or S6; Cell Signaling Technology), anti-phospho-eIF4E (Ser209; Cell Signaling Technology), anti-eIF4E (Cell Signaling Technology), anti-Bax (Cell Signaling Technology), anti-Bak (Cell Signaling Technology), anti-Bcl-2 (Cell Signaling Technology), anti-Ki-67 (Abcam), anti-Flag (Cell Signaling Technology), Normal rabbit IgG (Cell Signaling Technology), Alexa Fluor 488 goat anti-mouse IgG (Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen), anti-SLFN11 (Santa Cruz, sc-374339), and BrdU Cell Proliferation Assay Kit (Cell Signaling Technology). Cisplatin was purchased from MedChem Express. INK128 (also known as sapanisertib, MLN0128, and TAK-228) was purchased from SelleckChem.
Zhou C., Liu C., Liu W., Chen W., Yin Y., Li C.W., Hsu J.L., Sun J., Zhou Q., Li H., Hu B., Fu P., Atyah M., Ma Q., Xu Y., Dong Q., Hung M.C, & Ren N. (2020). SLFN11 inhibits hepatocellular carcinoma tumorigenesis and metastasis by targeting RPS4X via mTOR pathway. Theranostics, 10(10), 4627-4643.
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8

SCLC Cell Lines and Inhibitors

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MLN0128 and AZD1775 were purchased from Selleck Chemicals. Human SCLC cell lines NCI-H69 (H69), NCI-H82 (H82) and human embryonic kidney (293T/17) cells were purchased from ATCC. H69 and H82 were maintained in RPMI-1640 (ATCC) and 293T/17 cells were cultured in DMEM (Life Technologies). All media was supplemented with 10% FBS (Atlanta Biologicals), penicillin-streptomycin and L-glutamine (Sigma-Aldrich).
Fang B., Kannan A., Guo T, & Gao L. (2018). Simultaneously targeting DNA damage repair pathway and mTORC1/2 results in small cell lung cancer growth arrest via ER stress-induced apoptosis. International Journal of Biological Sciences, 14(10), 1221-1231.
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9

Inhibitors and Compounds Acquisition

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Testosterone was purchased from Sigma (Sigma Chemical, St. Louis, MO); Fulvestrant (ICI 182780; Faslodex) and everolimus (RAD-001) from Tocris (Ellisville, MO); and palbociclib (PD0332991) and MLN0128 (also known as TAK-228 or INK-128) from Selleckchem (Houston, TX). Inhibitors Erlotinib, Lapatinib, MK-2206 and PPP (Picropodophyllotoxin) were purchased from Cayman Chemical (Ann Arbor, MI).
Petrossian K., Nguyen D., Lo C., Kanaya N., Somlo G., Cui Y.X., Huang C.S, & Chen S. (2018). Use of dual mTOR inhibitor MLN0128 against everolimus-resistant breast cancer. Breast cancer research and treatment, 170(3), 499-506.
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