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Mrt67307

Manufactured by Merck Group
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MRT67307 is a lab equipment product manufactured by Merck Group. It is a small molecule that functions as a selective inhibitor. The core purpose of MRT67307 is to inhibit specific molecular targets in controlled laboratory settings.

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Lab products found in correlation

Bx795, by InvivoGen (3 mentions) Tpca 1, by Bio-Techne (3 mentions) Cycloheximide (chx), by Merck Group (3 mentions) Bx795, by Merck Group (3 mentions) Pf 3644022, by Bio-Techne (2 mentions) 7 oxozeanol, by Bio-Techne (2 mentions) Nec 1, by Abcam (2 mentions) Z vad fmk, by Abcam (2 mentions) Ethidium bromide, by Bio-Rad (2 mentions) Interferon γ, by BioLegend (2 mentions) Pepstatin a, by Merck Group (2 mentions) Bafilomycin a1, by Santa Cruz Biotechnology (2 mentions) Mitotempo, by Merck Group (2 mentions) 3 methyladenine 3 ma, by Merck Group (2 mentions) Oligomycin a, by Merck Group (2 mentions) Antimycin a, by Merck Group (2 mentions) Penicillin streptomycin, by Merck Group (2 mentions) Brefeldin a, by BD (2 mentions) Ly294002, by Cayman Chemical (2 mentions) Torin1, by Merck Group (2 mentions)

21 protocols using mrt67307

1

THP1 and MEF Cell Stimulation

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THP1 cells were treated with 500 ng/ml Ultrapure LPS from Escherichia coli 0111: B4 (Invivogen, Cat# tlrl-3pelps) or Salmonella minnesota (Invivogen, Cat# tlrl-smlps) in standard RPMI complete media. Similarly, THP1 cells in RMPI complete media were stimulated with 500 ng/ml Pam3CSK4 (Invivogen, Cat# tlrl-pms). Primary MEFs were transfected with 0.5 μg of 5′ppp-dsRNA or 4 μg HT-DNA per well of a 6-well plate using with Lipofectamine 2000 as described earlier117 (link). Dox (Sigma, Cat# D1515) was supplemented in the THP1 and MEF culture media at 0.5–2 μM. CPT (Cayman Cat# 11694) was used at a concentration of 1–5 μM for the indicated time periods. Cells were exposed to 5 Gy IR and harvested 1 h post-IR. THP1 cells were pretreated with 2 μM TBK1 inhibitor MRT67307 (EMD Millipore, Cat# 506306) or dimethyl sulfoxide vehicle control as described earlier118 (link) preceding cGAMP treatment. THP1 cells were stimulated with recombinant human IFN-β (0.5–100 ng/ml) (Peprotech, Cat# 300-02BC) in culture media. Cells were pretreated with 25 μM ATM inhibitor KU-55933 (Sigma Cat# SML1109), 10 μM PARP inhibitor olaparib (Cayman Cat# 10621), or 10 μM PARP1 inhibitor rucaparib (Cayman Cat# 15643) for 2 h. Cells were harvested at the indicated time points for analysis. Cellular NAD+ levels were boosted by preincubating cells for 24 h with 2–4 mM NAM (Sigma Cat# 47865-U).
Banerjee D., Langberg K., Abbas S., Odermatt E., Yerramothu P., Volaric M., Reidenbach M.A., Krentz K.J., Rubinstein C.D., Brautigan D.L., Abbas T., Gelfand B.D., Ambati J, & Kerur N. (2021). A non-canonical, interferon-independent signaling activity of cGAMP triggers DNA damage response signaling. Nature Communications, 12, 6207.
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2

Examination of Cell Death Mechanisms

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Birinapant and LCL-161 were purchased from MedChemExpress. zVAD-fmk was purchased from Bachem. Necrostatin-1 was obtained from Tocris Bioscience. MRT67307, TPCA-1 [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide and Protease Inhibitor Cocktail Set V were purchased from EMD Millipore.
Xu X., Kalac M., Markson M., Chan M., Brody J.D., Bhagat G., Ang R.L., Legarda D., Justus S.J., Liu F., Li Q., Xiong H, & Ting A.T. (2020). Reversal of CYLD phosphorylation as a novel therapeutic approach for adult T-cell leukemia/lymphoma (ATLL). Cell Death & Disease, 11(2), 94.
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3

Inhibiting Rab7 and TBK1/IKKε in STING Activation

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For all inhibitor studies, cells were pretreated for 30 min with each inhibitor +/− 1μg ml−1 poly(dA:dT) (Invivogen) prepared with X-tremeGENE™ 9. Rab7 was inhibited with 100nM of Ras Superfamily GTPases Binding Antagonist (CID 1067700, Millipore Sigma) and stimulated with poly(dA:dT) for 6 h prior to analysis. TBK1/IKKε inhibition immunofluorescence studies were performed with Compound 1 (Gilead Sciences), MRT67307 (Sigma-Aldrich), and CYT387 (MedChemExpress). Pretreatment or stimulation times were determined through a time course looking at STING perinuclear foci formation with 6 h, 8 h, and 3 h selected for each of the inhibitors respectively. Similarly, treatment times with the inhibitors were the same with the addition of poly(dA:dT).
Ritter J.L., Zhu Z., Thai T.C., Mahadevan N.R., Mertins P., Knelson E.H., Piel B.P., Han S., Jaffe J.D., Carr S.A., Barbie D.A, & Barbie T.U. (2019). Phosphorylation of RAB7 by TBK1/IKKε Regulates Innate Immune Signaling in Triple Negative Breast Cancer. Cancer research, 80(1), 44-56.
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4

Compound Screening for Cell Viability Assay

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Cells (2000/well) were seeded in a 96-well plate and treated with inhibitors at the indicated doses for 72 h prior to assessment of cell viability using Cell Titre Glo (Promega), following the manufacturer's recommendations. IC50 data were generated from dose-response curves fitted using a four-parameter regression fit in GraphPad Prism 6 software. Inhibitors used in this study include Gefitinib, Rociletinib, Lapatinib, Neratinib, Sorafenib, Ceritinib, Crizotinib, Pazopanib, Sunitinib, Dasatinib, Ponatinib, AZD4547, Bosutinib, BEZ235, Trametinib, NVP-AUY-922, Imatinib (LC laboratories) AZD9291, PF-562271, Palbociclib, BGJ398, MK2206, AZD5363 (Selleck Chemicals), BX-795, MRT67307 (Sigma-Aldrich), JQ1 (Cayman Chemical Company), DDR1-in-1 (Tocris), CCT244747 (ICR).
Vyse S., McCarthy F., Broncel M., Paul A., Wong J.P., Bhamra A, & Huang P.H. (2018). Quantitative phosphoproteomic analysis of acquired cancer drug resistance to pazopanib and dasatinib. Journal of Proteomics, 170, 130-140.
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5

Quantifying Cell Death Pathways

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Cell death was induced by exposure to ABT-737 (AbbVie), Dexamethasone (Sigma), WEHI-539 (MedChemExpress), or Etoposide (Hospira). Where indicated, cells were preincubated for 1 hr with MRT-67307 (Sigma) and for 15–30 min with ABT-737, followed by continuous exposure to Q-VD-Oph (SM Biochemicals) or zVAD.fmk (R&D Systems). Cell viability was quantified by CellTiterGlo (Promega) or flow cytometric analysis of cells excluding 5 µg/ml propidium iodide (PI) (Sigma) and, where indicated, cells also negative for AnnexinV-FITC (InvivoGen) binding using a FACSCailbur (BD) or LSRII (BD). Caspase activity was assayed by the addition of caspase3/7Glo (Promega) or by immunoblotting as described in the Extended Experimental Procedures.
White M.J., McArthur K., Metcalf D., Lane R.M., Cambier J.C., Herold M.J., van Delft M.F., Bedoui S., Lessene G., Ritchie M.E., Huang D.C, & Kile B.T. (2014). Apoptotic Caspases Suppress mtDNA-Induced STING-Mediated Type I IFN Production. Cell, 159(7), 1549-1562.
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6

Autophagy Inhibition Assay Reagents

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MG132 was purchased from Calbiochem (474790, San Diego, CA, USA). Autophagy-lysosome inhibitor chloroquine (CQ) was purchased from Sigma-Aldrich (C6628, St. Louis, MO, USA). Ammonium bicarbonate (1 mol/L, 012-21745), methanol (67-56-1), acetonitrile (012-19851) and formic acid (067-04531) for mass spectrometry were purchased from FUJIFILM Wako Pure Chemical Industries (Osaka, Japan). BX795 (S1274, InvivoGen, San Diego, CA, USA) and MRT67307 (SML0702, Sigma-Aldrich) were dissolved in dimethyl sulfoxide (DMSO).
Liu L., Matsumoto M., Watanabe-Matsui M., Nakagawa T., Nagasawa Y., Pang J., Callens B.K., Muto A., Ochiai K., Takekawa H., Alam M., Nishizawa H., Shirouzu M., Shima H., Nakayama K, & Igarashi K. (2024). TANK Binding Kinase 1 Promotes BACH1 Degradation through Both Phosphorylation-Dependent and -Independent Mechanisms without Relying on Heme and FBXO22. International Journal of Molecular Sciences, 25(8), 4141.
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7

Inhibitors for Cell Signaling Pathways

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The following inhibitors were used at the indicated final concentration in vitro, unless otherwise specified in the figure or figure legends: MRT-67307 (Sigma-Aldrich, 2 µM), BX-795 (Invivogen, 1 µM), TPCA-1 (Tocris Bioscience, 5 µM), PF-3644022 (Tocris Bioscience, 1 µM), 7-oxozeanol (Tocris Bioscience, 1 µM), Nec-1s (Biovision, 10 µM) zVAD-fmk (Abcam, 20 µM), cycloheximide (Sigma-Aldrich).
All antibodies used in this study are listed in supplementary table 7 including information regarding dilutions and validation.
Lafont E., Draber P., Rieser E., Reichert M., Kupka S., de Miguel D., Draberova H., von Mässenhausen A., Bhamra A., Henderson S., Wojdyla K., Chalk A., Surinova S., Linkermann A, & Walczak H. (2018). TBK1 and IKKε prevent TNF-induced cell death by RIPK1 phosphorylation. Nature cell biology, 20(12), 1389-1399.
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8

Inhibitors for Cell Signaling Pathways

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The following inhibitors were used at the indicated final concentration in vitro, unless otherwise specified in the figure or figure legends: MRT-67307 (Sigma-Aldrich, 2 µM), BX-795 (Invivogen, 1 µM), TPCA-1 (Tocris Bioscience, 5 µM), PF-3644022 (Tocris Bioscience, 1 µM), 7-oxozeanol (Tocris Bioscience, 1 µM), Nec-1s (Biovision, 10 µM) zVAD-fmk (Abcam, 20 µM), cycloheximide (Sigma-Aldrich).
All antibodies used in this study are listed in supplementary table 7 including information regarding dilutions and validation.
Lafont E., Draber P., Rieser E., Reichert M., Kupka S., de Miguel D., Draberova H., von Mässenhausen A., Bhamra A., Henderson S., Wojdyla K., Chalk A., Surinova S., Linkermann A, & Walczak H. (2018). TBK1 and IKKε prevent TNF-induced cell death by RIPK1 phosphorylation. Nature cell biology, 20(12), 1389-1399.
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9

Investigating ISG54 Promoter Activity Regulation

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For evaluation of ISG54 promoter activity, 2×105 WT or IRF3KO RAW-Lucia cells were incubated overnight in 500 μL cell culture media in 24-well plates. Cells were then incubated with fresh culture media containing PolyI:C (10 μg/mL) or IFN-γ (20 ng/mL), or a mixture of both. At 3, 6, 12, and 24 h, 10 μL of supernatant was removed, mixed with 40 μL of QuantiLuc ™ (InVivogen #repl-qlc1), after which luciferase luminescence was measured using a Turner Biosystems Luminometer, model TD-20/20. Alternatively, 1×105 WT or IRF3KO RAW-Cells were incubated in 200 μL cell culture media on 96-well plates and stimulated with agonists for TLR2, TLR4, TLR9, and STING. After 24 h, secreted luciferase was measured. To inhibit TBK1, 2×105 WT or IRF3KO RAW-Lucia cells were incubated overnight in 500 μL culture media in 24-well plates and then incubated with PBS or MRT67307 (2 μM) (Sigma Chem. Co.; SML0702) for 1 h followed by PBS, Poly I:C (25 μg/mL), IFN-γ (50 ng/mL), or both IFN-γ and Poly I:C. After 24 h, luciferase was measured as previously described.
Guinn Z.P, & Petro T.M. (2019). Interferon Regulatory Factor 3 plays a role in macrophage responses to Interferon-γ. Immunobiology, 224(4), 565-574.
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10

Isolation and Culture of MEFs and BMDMs

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MEFs were isolated from embryos at day 12.5-14.5 and maintained in culture for maximum 6 passages. BMDMs were differentiated from bone-marrow cells by using L929-supernatant (10%) enriched media for 6 days. Cells were grown in DMEM with 10% FBS (Atlanta Biological) and Penicillin/Streptomycin (Sigma). Interferon-γ (BioLegend #575306), Torin1 (EMD Millipore #475991), Brefeldin A (BD Biosciences #555029), MRT67307 (Sigma #CAS1190378-57-4), LY294002 (Cayman #70920), Bafilomycin A1 (Santa Cruz #sc-201550), E64d (Sigma #E8640), Pepstatin A (Sigma #P5318), 3-Methyladenine (3MA) (Sigma #M9281), MitoTEMPO (Sigma #SML0737), Oligomycin A (Sigma #75351), Antimycin A (Sigma #A8674), and Ethidium bromide (Bio-Rad #161-0433) were purchased.
Rai P., Janardhan K.S., Meacham J., Madenspacher J.H., Lin W.C., Karmaus P.W., Martinez J., Li Q.Z., Yan M., Zeng J., Grinstaff M.W., Shirihai O.S., Taylor G.A, & Fessler M.B. (2021). IRGM1 links mitochondrial quality control to autoimmunity. Nature immunology, 22(3), 312-321.
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