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Mrt68921

Manufactured by Selleck Chemicals
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MRT68921 is a compact and versatile lab equipment designed for various scientific applications. It features precise temperature control, reliable performance, and easy-to-use operation. The core function of MRT68921 is to provide a controlled environment for experiments and analysis.

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Lab products found in correlation

Sbi 0206965, by Selleck Chemicals (2 mentions) 3 methyladenine 3 ma, by Merck Group (2 mentions) Bafilomycin a1, by Merck Group (2 mentions) Chloroquine, by Selleck Chemicals (2 mentions) Antimycin a, by Merck Group (1 mentions) Wortmannin, by MedChemExpress (1 mentions) 3 methyladenine, by MedChemExpress (1 mentions) Eht 1864, by MedChemExpress (1 mentions) Pd 169 316, by MedChemExpress (1 mentions) Ehop 016, by MedChemExpress (1 mentions) Antibody against gapdh, by Proteintech (1 mentions) Z vad fmk, by R&D Systems (1 mentions) Gsk2606414, by Selleck Chemicals (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Ln229, by American Type Culture Collection (1 mentions) Torin2, by Merck Group (1 mentions) Gas pak pouches, by BD (1 mentions) Streptomycin, by Thermo Fisher Scientific (1 mentions) Penicillin, by Thermo Fisher Scientific (1 mentions) Rapamycin, by Merck Group (1 mentions)

12 protocols using mrt68921

1

Mitophagy Induction and ATG9A Regulation

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For translocation and mitophagy experiments, cells were either left untreated or treated with 10 mM Oligomycin (Calbiochem), 4 mM Antimycin A (Sigma) and 5 mM QVD (MedChemExpress) in full growth medium for different time points as indicated in figure legends. Long treatment time point samples were treated with 10 mM QVD instead of 5 mM. For starvation experiments, cells were fed in full medium for 1 h prior to 8 h starvation using Earle's Balanced Salt Solution (EBSS; Life Technologies). For the analysis of ATG9A (Figures 5A, 5D, S6A, and S6B), indicated samples were pre-treated for 30 min with 1 mM MRT68921 (Selleck Chemicals) before inducing mitophagy in the presence of 1 mM MRT68921.
Nguyen T.N., Padman B.S., Zellner S., Khuu G., Uoselis L., Lam W.K., Skulsuppaisarn M., Lindblom R.S.J., Watts E.M., Behrends C, & Lazarou M. (2021). ATG4 family proteins drive phagophore growth independently of the LC3/GABARAP lipidation system. Molecular cell, 81(9).
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2

Phospho-p38α and Phospho-MKK4 Antibody Protocol

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Antibodies against phospho-p38α (Thr180/Tyr182) (Cat# 4511S) and phospho-MKK4 (Ser257) (Cat# 4514S) were purchased from Cell Signaling Technologies (Beverly, MA, USA). Antibodies against MKK4 (Cat# A14781) were from ABclonal Technology (Wuhan, China). Antibody against p38 (Cat# sc-81621) was from Santa Cruz biotechnology (Santa Cruz, CA, USA). Antibody against GAPDH (Cat# 60004-1-Ig) was from Proteintech Group (Chicago, IA, USA). Tubeimoside-2 was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). Small-molecular inhibitor MRT68921 (Cat# S7949) was from Selleck Chemicals(Houston, TX, USA); other small molecular inhibitors, 3-methyladenine (Cat# HY-19312), Wortmannin (Cat# HY-10197), EIPA (Cat# HY-101840), PD 169,316 (Cat# HY-10578), EHop-016 (Cat# HY-12810), and EHT 1864 (Cat# HY-16659) were purchased from MedChemExpress (MCE, Shanghai, China). We purchased 70 kDa dextran (Cat# D1818) and lucifer yellow (#L1177) from Life Technologies (Waltham, MA, USA). All reagents unless otherwise stated were purchased from Thermo Fisher Scientific (Waltham, MA, USA).
Gan Y., Wang C., Chen Y., Hua L., Fang H., Li S., Chai S., Xu Y., Zhang J, & Gu Y. (2023). Tubeimoside-2 Triggers Methuosis in Hepatocarcinoma Cells through the MKK4–p38α Axis. Pharmaceutics, 15(4), 1093.
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3

Autophagy Modulation with Pharmacological Inhibitors

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Stock solutions of reagents were prepared by dissolving the reagents in dimethyl sulfoxide (DMSO, Sigma-Aldrich). SBI-0206965, GSK 2606414, and MRT 68921 were purchased from Selleck Chemicals (Houston, TX, USA). Bafilomycin A1 and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich. Hydroxychloroquine (HCQ) was purchased from Myung-in Pharmaceuticals. z-VAD-FMK was obtained from R&D Systems. Control cells were treated with equal amounts of DMSO.
Hwang D.Y., Eom J.I., Jang J.E., Jeung H.K., Chung H., Kim J.S., Cheong J.W, & Min Y.H. (2020). ULK1 inhibition as a targeted therapeutic strategy for FLT3-ITD-mutated acute myeloid leukemia. Journal of Experimental & Clinical Cancer Research : CR, 39, 85.
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4

Glioblastoma Cell Culture Optimization

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Reagents included torin2 and rapamycin (both Sigma-Aldrich, St. Louis, MO, USA), bafilomycin A1 (Cayman Chemicals, Ann Arbor, MI, USA) and MRT68921 (Selleck Chemicals, Houston, TX, USA). LN-229 were purchased from ATCC (Manassas, Virginia, USA). LN-308 cells were a kind gift from Prof. Nicolas de Tribolet (Lausanne, Switzerland) and were recently authenticated by short tandem repeat (STR) profiling. Cells were maintained at 37 °C and 5% CO2 using Dulbecco’s modified eagle medium (DMEM). The medium was supplemented with 10% fetal calf serum (Biochrom KG, Berlin, Germany), 100 µg/mL streptomycin and 100 IU/mL penicillin (Life Technologies, Karlsruhe, Germany). Cells were tested regularly for mycoplasma infection. For starvation conditions, cells were incubated in serum-free medium with 2 mM glucose. For hypoxic experimental conditions of 0.1% oxygen, we used Gas Pak pouches (Becton-Dickinson, Heidelberg, Germany).
Divé I., Klann K., Michaelis J.B., Heinzen D., Steinbach J.P., Münch C, & Ronellenfitsch M.W. (2022). Inhibition of mTOR signaling protects human glioma cells from hypoxia-induced cell death in an autophagy-independent manner. Cell Death Discovery, 8, 409.
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5

Cancer Cell Line Cultivation Protocol

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The human cancer cell lines A549, H1299, NCI-H460, MNK45, U251, SW480, SW620, HCT116, Colo320 and HT-29, PC-3, U266, and the mouse breast cancer cell line 4T1 were cryopreserved in the Hematological Laboratory of Zhujiang Hospital (Guangzhou, China). All cell lines were incubated in DMEM medium supplemented with 10% fetal bovine serum at 37 °C with 5% CO2. WZ4003, SBI-0206965, Chloroquine, and MRT68921 were purchased from Selleckchem (Houston, TX, USA), dissolved in DMSO or water, and stored at −20 °C. CCK-8 was purchased from Dojindo Laboratories (Japan). Mitotracker, DAPI, and TritonX-100 were purchased from Solarbio (Beijing, China).
Chen Y., Xie X., Wang C., Hu Y., Zhang H., Zhang L., Tu S., He Y, & Li Y. (2020). Dual targeting of NUAK1 and ULK1 using the multitargeted inhibitor MRT68921 exerts potent antitumor activities. Cell Death & Disease, 11(8), 712.
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6

Western Blot Antibody Validation Protocol

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Primary antibodies used in Western blot studies included anti-LC3B antibody (#L7543, 1:1000) from Sigma-Aldrich (Burlington, MA, USA), anti-MYC (Y69, #ab32072, 1:1000) from Abcam (Cambridge, MA, USA), anti-ULK1 (D8H5, #8054, 1:1000) from Cell Signaling Technology (Danvers, MA, USA), anti-ULK2 (#PA5-22173, 1:1000) from ThermoFisher Scientific (Waltham, MA, USA), and anti-β-actin (#sc-47778, 1:2000) from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MRT68921 (#S7949), Ara-C (U-19920A, #S1648), and chloroquine (#S6999) were purchased from Selleckchem (Houston, TX, USA). IRDye 800CW goat anti-rabbit IgG (#926-32213, 1:40,000) and anti-mouse IgG (#926-32212, 1:40,000) were used as the secondary antibody (LI-COR Biosciences, Lincoln, NE, USA).
Lai J., Yang C., Shang C., Chen W., Chu M.P., Brandwein J., Lai R, & Wang P. (2024). ULK2 Is a Key Pro-Autophagy Protein That Contributes to the High Chemoresistance and Disease Relapse in FLT3-Mutated Acute Myeloid Leukemia. International Journal of Molecular Sciences, 25(1), 646.
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7

Autophagy Modulation by Small Molecules

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Forskolin, doxorubicin, propidium iodide (PI), paraformaldehyde (PFA), and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich. 8-CPT-cAMP and RP-8-Br-cAMPS were from BioLog, and bafilomycin A1 (BafA1) was from AH Diagnostics. The ULK1 inhibitor MRT68921 was obtained from Selleckchem. Antibody against p53 (DO-1, # SC-126) was obtained from Santa Cruz Biotechnology. Anti-LC3B (# 2775) used for Western blot analyses was purchased from Cell Signaling Technology, whereas Anti-LC3B (# PM036) used for immunofluorescence analyses was obtained from MBL International. Antibodies against glyceralaldehyde 3-phosphate dehydrogenase (GAPDH) was purchased from Sigma Aldrich. As loading control in Western blot analyses we used an antibody directed against Calnexin (# 2433) from Cell Signaling Technology.
Skah S., Richartz N., Duthil E., Gilljam K.M., Bindesbøll C., Naderi E.H., Eriksen A.B., Ruud E., Dirdal M.M., Simonsen A, & Blomhoff H.K. (2018). cAMP-mediated autophagy inhibits DNA damage-induced death of leukemia cells independent of p53. Oncotarget, 9(54), 30434-30449.
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8

Lipid Reagents for Cellular Assays

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All lipids were purchased pre-dissolved in chloroform, except for PI(3)P, from Avanti Polar Lipids. Lipids used in Fig. 1 and Supplementary Fig. 1 are: L-α-phosphatidylethanolamine (840022C) (BrainPE), L-α-phosphatidylserine (840032C) (Brain PS) and L-α-phosphatidylcholine (840053C) (Brain PC). Lipids used in Fig. 2, 3 and 6 are: 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (850725C) (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (810150C) (Rhod-PE), 1palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (850457C) (POPC), 1,2-dioleoyl-sn-glycero3-phospho-(1’-myo-inositol-3’-phosphate) (850150P) (PI(3)P) and L-α-phosphatidylinositol (Liver, Bovine) (840042C) (PI). Adenosine-5’-triphosphate (A2383, Sigma) (ATP), Bafilomycin A1 (BML-CM110, AH diagnostics) (BafA1), Deferiprone (379409, Sigma) (DFP), Fibronectin (F2006–1MG, Sigma), Hoechst 33342 (H1399, Thermo Fisher Scientific), Hygromycin b (400052–20), Monensin sodium salt (M5273, Sigma), MRT68921 (S7949, Selleckchem), Puromycin dihydrochloride (P7255, Sigma), VPS34-IN1 (S7980, Selleckchem), Zeocin (45–0430, Invitrogen), Zymosan A particles (Alexa 594 Z23374 and Alexa 488 Z23373, Thermo Fischer Scientific).
Lystad A.H., Carlsson S.R., de la Ballina L.R., Kauffman K.J., Nag S., Yoshimori T., Melia J.T, & Simonsen A. (2019). Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes. Nature cell biology, 21(3), 372-383.
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9

Combinatorial Drug Screening in Cancer Cells

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Cells were plated in 96-well plates and exposed to AZD5991 (Chemietek Cat#CT-A5991 or Astrazeneca), AMG176 (MedChemExpress), AZD4320 (Astrazeneca), Venetoclax (Selleckchem), Azacytidine (Selleckchem), Venetoclax+Azacytidine (Ven:Aza = 1:4), Chloroquine (Sigma Aldrich, Cat#C6628) or ULK1i (MRT68921, Selleckchem, Cat#S7949) with a minimum of three technical replicates per concentration per cell line. Cell viability was measured with the CellTiter-Glo 2.0 reagent (Promega, Cat#G9243) according to manufacturer’s instructions. Absolute viability values were converted to percentage viability versus DMSO control treatment, and then non-linear fit of log(inhibitor) versus response (three parameters) was performed in GraphPad Prism v8.0 to obtain the IC50 values. For multi-drug combination profiling data, SynergyFinder (version 2.0) was used (https://synergyfinder.fimm.fi/). The expected drug combination responses were calculated based on ZIP reference model using SynergyFinder83 (link). Deviations between observed and expected responses with positive and negative values denote synergy and antagonism respectively. For estimation of outlier measurements, cNMF algorithm84 (link) implemented in SynergyFinder was utilized.
Gradinaru C.C., Kennis J.T., Papagiannakis E., van Stokkum I.H., Cogdell R.J., Fleming G.R., Niederman R.A, & van Grondelle R. (2001). An unusual pathway of excitation energy deactivation in carotenoids: Singlet-to-triplet conversion on an ultrafast timescale in a photosynthetic antenna. Proceedings of the National Academy of Sciences of the United States of America, 98(5), 2364-2369.
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10

Probing Signaling Pathways via Western Blot and IP

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Western blotting was performed as described previously [63 (link)]. Briefly, cell or tissue samples were separated on SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane, and probed with antibodies as indicated. Immunoprecipitation (IP) was performed as previously described [59 (link)]. RNA immunoprecipitation (RIP) was performed using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Burlington, MA, USA) according to the manufacturer’s instructions. The following antibodies were used in this study: ATG7 (Proteintech, Wuhan, China), IRF3 (Cell Signaling Technology, Danvers, MA, USA), phospho-IRF3 (S386) (abcam, Cambridge, UK), P65 (Cell Signaling Technology, Danvers, MA, USA), phospho-P65 (S536) (Cell Signaling Technology, Danvers, MA, USA), and actin (Santa Cruz Biotechnology, Dallas, TX, USA). The antibody against IAV NP was generated and used as previously described [58 (link)]. The following reagents were used in this study: dimethyl sulfoxide (DMSO) (Sigma-Aldrich), hydroxychloroquine Sulfate (HCQ) (Selleck, Houston, TX, USA), and MRT68921 (Selleck).
Chen B., Guo G., Wang G., Zhu Q., Wang L., Shi W., Wang S., Chen Y., Chi X., Wen F., Maarouf M., Huang S., Yang Z, & Chen J.L. (2024). ATG7/GAPLINC/IRF3 axis plays a critical role in regulating pathogenesis of influenza A virus. PLOS Pathogens, 20(1), e1011958.
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