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Tunicamycin

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Tunicamycin is a laboratory reagent used in cell biology research. It functions as a potent inhibitor of N-linked glycosylation, a critical process in the biosynthesis of glycoproteins within eukaryotic cells.

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Lab products found in correlation

Thapsigargin, by Bio-Techne (5 mentions) Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions) Carfilzomib, by Apexbio (1 mentions) Dithiothreitol (dtt), by Thermo Fisher Scientific (1 mentions) Rapamycin, by InvivoGen (1 mentions) Ionomycin, by Bio-Techne (1 mentions) Bafilomycin a1, by InvivoGen (1 mentions) Sodium arsenite solution, by Merck Group (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions) Z ietd fmk, by R&D Systems (1 mentions) Z lehd fmk, by R&D Systems (1 mentions) Z vad fmk, by InvivoGen (1 mentions) Gsk2606414, by MedChemExpress (1 mentions) Gsk2656157, by MedChemExpress (1 mentions) Amg perk 44, by Bio-Techne (1 mentions) Etoposide, by Merck Group (1 mentions) Ht501128, by Merck Group (1 mentions) Penn strep, by Thermo Fisher Scientific (1 mentions) Pcmv myc, by Takara Bio (1 mentions) Pcmv flag, by Merck Group (1 mentions)

20 protocols using tunicamycin

1

Mycolactone Purification and Storage

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Mycolactone was purified from M. ulcerans bacterial pellets (strain 1615, ATCC 35840), then quantified by spectrophotometry, and stored in ethanol at −20°C protected from light. For in vivo experiments, a 4 mM stock was diluted in a NaCl solution (0.9% w/v) immediately before injection in animals. For in vitro experiments, a 1,000× working solution was prepared by dilution of the ethanol stock in DMSO and stored at −20°C, then thawed and diluted in culture medium immediately before use. BZ purchased from Alfa Aesar (#J60378) was resuspended in DMSO to yield a 10 mM solution stored at −20°C. A 10 mM solution of carfilzomib in DMSO was purchased from ApexBio (#A1933) and stored at −20°C. BZ and carfilzomib were thawed and diluted in culture medium immediately before use. Thapsigargin (Tocris, #1138) and tunicamycin (Tocris, #3516) were resuspended in DMSO to yield a 10 mM solution stored at −20°C; and dithiothreitol (DTT, 1 M solution) was purchased from Invitrogen (#P2325) and stored at −20°C.
Domenger A., Choisy C., Baron L., Mayau V., Perthame E., Deriano L., Arnulf B., Bories J., Dadaglio G, & Demangel C. (2022). The Sec61 translocon is a therapeutic vulnerability in multiple myeloma. EMBO Molecular Medicine, 14(3), e14740.
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2

siRNA Knockdown of STAU1 and mTOR

Cited 2 times
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The small interfering RNAs (siRNAs) used in this study were: All Star Negative Control siRNA (QIAGEN, Hilden, Germany; Cat# 1027280), human siSTAU1: 5′-CCUAUAACUACAACAUGAGdTdT-3′,11 (link),15 (link)–17 (link) and siMTOR: 5′-GAG CCUUGUUGAUCCUUAA-3′.19 (link) All siRNA oligonucleotides were synthesized by Invitrogen (Carlsbad, CA). The oligonucleotides were deprotected, and the complementary strands were annealed. Bafilomycin A1 (Baf; InvivoGen, San Diego, CA; Cat# tlrl-baf1), rapamycin (InvivoGen, Cat# tlrl-rap), thapsigargin (Tocris, Ellisville, MO; Cat# 1138), tunicamycin (Tocris, Cat# 3516), Ionomycin (Tocris, Cat# 1704), and sodium arsenite solution (Sigma-Aldrich, St Louis, MO; Cat# 1062771000) were used in this study.
Paul S., Dansithong W., Gandelman M., Figueroa K.P., Zu T., Ranum L.P., Scoles D.R, & Pulst S.M. (2022). Staufen Impairs Autophagy in Neurodegeneration. Annals of neurology, 93(2), 398-416.
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3

Inducing APOL1 expression in kidney organoids

Cited 2 times
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Day 24 G0 and G1 kidney organoids in identically plated wells of a 24-well plate were treated with IFN-γ (25 ng/ml; PeproTech) for 24 hours to induce APOL1 expression. This dose approximates previous in vitro IFN-γ doses used for macrophage activation (22 (link),23 (link)). Endoplasmic reticulum (ER) stress was induced by adding 5 μM tunicamycin (Tocris) for 24 hours.
Liu E., Radmanesh B., Chung B.H., Donnan M.D., Yi D., Dadi A., Smith K.D., Himmelfarb J., Li M., Freedman B.S, & Lin J. (2020). Profiling APOL1 Nephropathy Risk Variants in Genome-Edited Kidney Organoids with Single-Cell Transcriptomics. Kidney360, 1(3), 203-215.
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4

ER Stress Induction and PERK Inhibition

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MCF7 cells were purchased from ECACC. Wild type and NCOA3 knockout HeLa cells were a kind gift from Bert O’Malley, Baylor College of Medicine, USA. HEK 293T cells were from Indiana University National Gene Vector Biorepository. Cells maintained in Dulbecco’s modified medium (DMEM) supplemented with 10% FBS, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C with 5% CO2. To induce ER stress, cells were treated with tunicamycin (TM), Thapsigargin (TG) or Brefeldin A (BFA) at the indicated concentrations for the indicated time. Thapsigargin (Cat # 1138), tunicamycin (Cat # 3516), BFA (Cat # 1231) were sourced from Tocris Bioscience. MCB-613 (SML1567) was obtained from Sigma-Aldrich, Ireland. To inhibit PERK activity cells were treated with GSK2606414 (Cat # 516535 Millipore Ireland B.V).
Hossain M.M., Barua D., Arabkari V., Islam N., Gupta A, & Gupta S. (2018). Hyperactivation of nuclear receptor coactivators induces PERK-dependent cell death. Oncotarget, 9(14), 11707-11721.
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5

Pharmacological Inhibitors of UPR Pathways

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Selective PERK inhibitors GSK2656157 and GSK2606414 were obtained from MedChemExpress, and AMG PERK 44 was obtained from Tocris Bioscience. The IRE1α inhibitor 4μ8c was obtained from Sigma-Aldrich. The pan-caspase inhibitor z-VAD-fmk was obtained from Invivogen. The caspase-8 inhibitor z-IETD-fmk and the caspase-9 inhibitor z-LEHD-fmk were purchased from R&D Systems. The apoptosis enhancer etoposide was obtained from Sigma-Aldrich. The UPR inducer tunicamycin was obtained from Tocris Bioscience.
Chu H., Shuai H., Hou Y., Zhang X., Wen L., Huang X., Hu B., Yang D., Wang Y., Yoon C., Wong B.H., Li C., Zhao X., Poon V.K., Cai J.P., Wong K.K., Yeung M.L., Zhou J., Au-Yeung R.K., Yuan S., Jin D.Y., Kok K.H., Perlman S., Chan J.F, & Yuen K.Y. (2021). Targeting highly pathogenic coronavirus-induced apoptosis reduces viral pathogenesis and disease severity. Science Advances, 7(25), eabf8577.
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6

Kidney Slice Culture for ER Stress

Cited 2 times
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A modified version of a previously published protocol was used49 . Human kidney pieces were decapsulated and embedded in 3% agarose prewarmed at 40 °C. Slices were cut in ice-cold DPBS using a vibrating microtome Compresstome® VF310-0Z (Precisionary Instruments). Immediately after, the 300-μm-thick slices were pre-incubated containing Williams’ Medium E + GlutaMAX™-I (Gibco™, ThermoFisher Scientific) supplemented with 25 mM D-glucose and 1% Penn-Strep (10,000 U/mL, Gibco™, ThermoFisher Scientific) for 1 h at 37 °C under 80% O2 and 5% CO2 atmosphere to allow the slices to restore their ATP levels71 (link). Slices were then cultured for an additional 24 h with 1 μM tunicamycin72 (link) (#3516, Tocris Bioscience) and 0.05 μg/mL recombinant human Klotho (#5334-KL, R&D Systems) at 37 °C, 80% O2 and 5% CO2 on an orbital shaker at 80 RPM. Slices were harvested in 10% formalin solution (HT501128, Sigma-Aldrich) for histology analysis and in RNAlater for RNA isolation. All conditions were carried out as three technical replicates.
Charrin E., Dabaghie D., Sen I., Unnersjö-Jess D., Möller-Hackbarth K., Burmakin M., Mencke R., Zambrano S., Patrakka J, & Olauson H. (2023). Soluble Klotho protects against glomerular injury through regulation of ER stress response. Communications Biology, 6, 208.
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7

Characterization of HRD1 and METTL14 Mutants

Cited 1 time
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The full-length and truncation mutants of HRD1, HA-Ubiquitin expression plasmid, Flag-METTL14 expression were used as reported previously (Kong et al., 2016 (link); Yang et al., 2014 (link)). Myc-METTL14 was constructed to pCMV-Myc (Clontech, CA, USA) as reported before (Kong et al., 2016 (link); Yang et al., 2014 (link)). The truncation mutants of both HRD1 and METTL14 were generated by PCR and subcloned into pCMV-Flag (Sigma-Aldrich) or pCMV-Myc vectors (Clontech, CA, USA). Antibodies to Flag (F-1804), Anti-Actin (A2103), Anti-HRD1 (HPA024300) were purchased from Sigma (St. Louis, MO, USA), and antibodies to Myc (sc-40), Myc-HRP (sc-40-HRP) and HA-HRP (sc-7392), Anti-GFP (sc-8334) were purchased from Santa Cruz (Santa Cruz, CA, USA). Anti-METTL14 (ab98166), anti-METTL3 (ab195352), anti-FTO (ab124892) and anti-ALKBH5 (ab195377) anti-human AAT (EPR17087-50) were purchased from Abcam (Cambridge, MA, USA). Anti-GAPDH (2118S), Anti-Ubiquitin (3933S), Anti-CHOP (5554S) were purchased from Cell Signaling Technology (Danvers, MA, USA). Tunicamycin was purchase from Tocris. Poly I:C, Actinomycin D and cycloheximide (CHX) were purchase from Sigma (St. Louis, MO, USA)
Wei J., Harada B.T., Lu D., Ma R., Gao B., Xu Y., Montauti E., Mani N., Chaudhuri S., Gregory S., Weinberg S., Zhang D., Green R., He C, & Fang D. (2021). HRD1-mediated METTL14 degradation regulates m6A mRNA modification to suppress ER proteotoxic liver disease. Molecular cell, 81(24), 5052-5065.e6.
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8

Inhibition of IRE1, DAPK1, and Pin1

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The IRE1 RNase inhibitor 4μ8c was purchased from EMD Millipore (catalog no. 412512), ER Stress Inducer Tunicamycin was purchased from Tocris (catalog no. 3516), potent and selective, ATP-competitive inhibitor of death-associated protein kinase 1 (DAPK1) TC-DAPK 6 (catalog no. 4301). Pin1 inhibitor Sulfopin (PIN1-3), which is a highly selective covalent inhibitor of Pin1was purchased from Selleckchem (Catalog No.S9782).
Jash S., Banerjee S., Cheng S., Wang B., Qiu C., Kondo A., Ernerudh J., Zhou X.Z., Lu K.P, & Sharma S. (2023). Cis P-tau is a central circulating and placental etiologic driver and therapeutic target of preeclampsia. Nature Communications, 14, 5414.
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9

Pharmacological Agents in Biomedical Research

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Phenylephrine hydrochloride, acetylcholine, NADPH, angiotensin II, and metformin were obtained from Sigma–Aldrich (USA). U46619 and tunicamycin were obtained from Tocris Bioscience. Stock solutions of drugs were prepared in ultrapure water and stored at −20 °C, and appropriate dilutions were made on the day of the experiment.
Chen C., Kassan A., Castañeda D., Gabani M., Choi S.K, & Kassan M. (2019). Metformin prevents vascular damage in hypertension through the AMPK/ER stress pathway. Hypertension research : official journal of the Japanese Society of Hypertension, 42(7), 960-969.
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10

Modulation of Cellular Stress Pathways

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Tunicamycin (cat. 3516) and salubrinal (cat. 2347) were from Tocris. Rapamycin (cat. HY-10219) was from MedChemExpress. All screened compounds were provided by Janssen Pharmaceuticals.
Lebedeva I.V., Wagner M.V., Sahdeo S., Lu Y.F., Anyanwu-Ofili A., Harms M.B., Wadia J.S., Rajagopal G., Boland M.J, & Goldstein D.B. (2021). Precision genetic cellular models identify therapies protective against ER stress. Cell Death & Disease, 12(8), 770.
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