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Harringtonine

Manufactured by LKT Laboratories
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Sourced in Japan

Harringtonine is a natural compound extracted from the Chinese evergreen tree Cephalotaxus harringtonia. It is a chemical reagent used for research purposes in laboratory settings. The primary function of Harringtonine is to inhibit protein synthesis in eukaryotic cells.

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Lab products found in correlation

Cycloheximide (chx), by Merck Group (3 mentions) Torin1, by Bio-Techne (2 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions) Penicillin streptomycin glutamine (psg), by Thermo Fisher Scientific (2 mentions) Harringtonine, by Merck Group (1 mentions) Hiseq 2000 system, by Illumina (1 mentions) Ribo zero gold rrna removal kit, by Illumina (1 mentions) Rnaimax, by Thermo Fisher Scientific (1 mentions) Cycloheximide (chx), by LKT Laboratories (1 mentions) Ribo zero human mouse rat kit, by Illumina (1 mentions) Nextseq 500 platform, by Illumina (1 mentions) Em 1 econo uv monitor, by Bio-Rad (1 mentions) Sorbitol, by Merck Group (1 mentions) Sephin1, by Apexbio (1 mentions) Sal003, by Bio-Techne (1 mentions) Salubrinal, by Santa Cruz Biotechnology (1 mentions) T 2 toxin, by Cayman Chemical (1 mentions) Spermidine trihydrochloride, by Merck Group (1 mentions) Tbe urea gel, by Thermo Fisher Scientific (1 mentions) Nebnext small rna library preparation kit, by New England Biolabs (1 mentions)

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Harringtonine (HT) (2 citations)

7 protocols using harringtonine

1

Investigating ER Stress Response Pathways

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For Ire1, Xbp1, and Perk siRNA experiments, we cultured cells in antibiotic-free media. We followed published RNAiMAX (Invitrogen) protocols to transfect cells with organism-specific siRNAs (Qiagen, Valencia, CA). The following siRNA reagents were used: Ire1 (SI00995890, 897, 904), Xbp1 (GS22433), Perk combined (GS1366-mouse or GS9451-human), Perk #1 (SI00991319), and Perk #3 (SI00991333). We controlled for the effects of the siRNA procedure by including Neg siRNA (Qiagen)–transfected samples in all experiments. We incubated cells for 48–72 h before replacing media and treating with or without ER stress. For harringtonine experiments, we added 1 μg/ml harringtonine (LKT Laboratories, St. Paul, MN) to media in Neg and Perk siRNA–treated cells and incubated for ∼5 min before the addition of DTT.
Moore K, & Hollien J. (2015). Ire1-mediated decay in mammalian cells relies on mRNA sequence, structure, and translational status. Molecular Biology of the Cell, 26(16), 2873-2884.
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2

Ribosome Profiling of Infected BMMs

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Ribosome profiling experiments were undertaken as previously described (Ingolia et al., 2012 (link)). BMMs were plated in tissue culture treated six-well plates (1.5 × 106 BMMs/well) or 75 cm2 flasks (1.2 × 107 BMMs per flask). At 6 hr post-infection BMMs were lysed by flash freezing and thawed in the presence of lysis buffer (Ingolia et al., 2012 (link)). When used, harringtonine (LKT Laboratories, Saint Paul, MN) was added at a final concentration of 2 μg/mL for 120 s at the end of the 6 hr infection. 100 μg/mL of cycloheximide (Sigma-Aldrich, St. Louis, MO) was added to freeze ribosomes after the 120 s harringtonine treatment. Following cycloheximide treatment cells were immediately lysed. Clarified lysates were split and some was used to generate ribosome footprints while some was used to isolate total RNA for RNA sequencing (described below). All RNA and DNA gel extractions were performed overnight as previously described (Ingolia et al., 2012 (link)). The Ribo-Zero Gold rRNA Removal Kit (Illumina, San Diego, CA) was used to remove rRNA from ribosome profiling samples before the dephosphorylation and linker ligation steps (Ingolia et al., 2012 (link)). Final ribosome profiling libraries were sequenced on a HiSeq2000 System (Illumina) with single read 50 (SR50) read lengths by the Vincent J. Coates Genomics Sequencing Laboratory at UC, Berkeley.
Barry K.C., Ingolia N.T, & Vance R.E. (2017). Global analysis of gene expression reveals mRNA superinduction is required for the inducible immune response to a bacterial pathogen. eLife, 6, e22707.
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3

Profiling of Ribosome-Associated Transcripts in EToV-Infected Cells

Cited 2 times
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ED cells were infected with EToV for 1 h in serum-free medium (MOI of 0.1) and flash-frozen in liquid nitrogen at 8 h.p.i. prior to either RNA isolation or ribosome purification for profiling. Cells were either not pretreated or, where stated, were treated with a final concentration of 100 μg/ml cycloheximide (CHX) for 2 min (Sigma-Aldrich) or 2 μg/ml of harringtonine for 3 min (LKT Laboratories), followed by CHX for 2 min before flash-freezing. RNA and ribosomes were harvested according to previously published protocols (17 (link), 58 (link)), with minor modifications. Following either RPF or RNA isolation, duplex-specific nuclease was not utilized but instead rRNA was depleted with the RiboZero [human/mouse/rat] kit (Illumina). Libraries were prepared and sequenced using the NextSeq500 platform (Illumina).
Stewart H., Brown K., Dinan A.M., Irigoyen N., Snijder E.J, & Firth A.E. (2018). Transcriptional and Translational Landscape of Equine Torovirus. Journal of Virology, 92(17), e00589-18.
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4

Ribosome Profiling of Drug-Treated Cells

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HeLa cells grown to 80% confluence in 10-cm dishes were treated with DMSO or 4 (5 μM) for 20 min (Figure 2c, left panel), or were first treated with DMSO, 4 (5 μM), or cycloheximide (CHX, 100 μg/mL, Sigma, St. Louis, MO) for 15 min, followed by harringtonine (2 μg/mL, LKT Laboratories, St. Paul, MN) for 20 min. Following treatment, dishes were placed on ice, media aspirated, and cells were washed once with ice-cold PBS. Cells were scraped into 400 μL lysis buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1% Triton X-100, 100 μg/mL CHX). Lysates were incubated for 10 min on ice, then passed through a 26-G needle 5 times. Samples were clarified by centrifugation at 14,000 rpm for 10 min at 4°C. Lysates were applied to 10–50% sucrose gradients (prepared in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 μg/mL CHX), followed by centrifugation at 35,000 rpm for 3 hr in a SW41 rotor at 4°C. Ribosome/polysome profiles were obtained by flowing the gradient top to bottom through an in-line spectrometer (254 nm, EM-1 Econo UV Monitor, BioRad, Hercules, CA).
Carelli J.D., Sethofer S.G., Smith G.A., Miller H.R., Simard J.L., Merrick W.C., Jain R.K., Ross N.T, & Taunton J. (2015). Ternatin and improved synthetic variants kill cancer cells by targeting the elongation factor-1A ternary complex. eLife, 4, e10222.
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5

Induction of Cellular Stress Responses

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The MEFs used were described previously (Scheuner et al., 2001 (link)). The cells were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific, 11960044) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, 26140079), 1X Penicillin-Streptomycin-Glutamine (Thermo Fisher Scientific, 10378016) at 37°C and 5% CO2. For all experiments, cells were subcultured 24 hours prior to the experiment so that cells were ~80% confluent at the time of the experiment. GADD34−/− MEFs were a gift from D. Ron (Novoa et al., 2003 (link)). Hyperosmotic stress was induced with Sorbitol (Sigma-Aldrich, S1876). Other chemicals used in this study include Cycloheximide (CHX) (100 μg/mL) (Sigma-Aldrich C7698), Harringtonine (2 μg/mL) (LKT Laboratories, H0169), Sephin1 (Apexbio, A8708), Sal 003 (1 μM) (Tocris Bioscience, 3657), Torin 1 (250 nM) (Tocris Bioscience, 4247), Hippuristanol (1 μM) (a generous gift from Dr. Junichi Tanaka, University of the Ryukyus, Japan) (Bordeleau et al., 2006 (link)) and CCCP (10 μM) (Sigma-Aldrich, C2759).
Jobava R., Mao Y., Guan B.J., Hu D., Krokowski D., Chen C.W., Shu X.E., Chukwurah E., Wu J., Gao Z., Zagore L.L., Merrick W.C., Trifunovic A., Hsieh A.C., Valadkhan S., Zhang Y., Qi X., Jankowsky E., Topisirovic I., Licatalosi D.D., Qian S.B, & Hatzoglou M. (2021). Adaptive translational pausing is a hallmark of the cellular response to severe environmental stress. Molecular cell, 81(20), 4191-4208.e8.
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6

Reagents for Cell Stress Assays

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The following reagents were used in this study: torin1 (Tocris Bioscience), harringtonine (LKT Laboratories), T-2 toxin (Cayman), spermidine trihydrochloride (Sigma-Aldrich), salubrinal and NSC119889 (Santa Cruz Biotechnology). Sodium ortho-arsenite (in the form of dihydroarsenite, NaH2AsO3) was kindly provided by Pavel Ivanov (MSU). 10 mM harringtonine, T-2 toxin, salubrinal and NSC119889 stock solutions in DMSO, 100 μM torin1 in DMSO, 1 M spermidine and arsenite water solutions were prepared and stored at −85 °C.
Akulich K.A., Andreev D.E., Terenin I.M., Smirnova V.V., Anisimova A.S., Makeeva D.S., Arkhipova V.I., Stolboushkina E.A., Garber M.B., Prokofjeva M.M., Spirin P.V., Prassolov V.S., Shatsky I.N, & Dmitriev S.E. (2016). Four translation initiation pathways employed by the leaderless mRNA in eukaryotes. Scientific Reports, 6, 37905.
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7

Ribosome Profiling of Embryonic Development

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Polysome profiling was performed on all six samples. Briefly, embryos from each time period (i.e., sample) were treated with harringtonine (LKT Laboratories, H0169) in mild lysis buffer followed by the addition of cycloheximide and immediate grinding. Samples were subjected to a 10%–50% sucrose gradient and all polysomes were collected and combined. Collected polysome fractions were pelleted via sucrose cushion (34%) and subjected to RNaseI digestion. After resuspension another cushion (34%) was performed and RNA was coprecipitated with GlycoBlue (Thermo Fisher, cat. No. AM95150). Recovered RNA was then run on a 15% TBE-Urea Gel (Thermo Fisher cat. no. EC68852BOX), followed by gel size selection to isolate ribosome protected fragments (26–31 nt) (ZR small-RNA PAGE Recovery Kit, Zymo Research, cat. no. R1070). Following end-repair phosphorylation of RNA molecules, libraries were constructed with the NEBNext Small RNA Library Preparation Kit according to manufacturer’s recommendations (NEB, cat. no. E7330). See Supplemental File 6 for step-by-step protocol.
Bunin B.A., Plunkett M.J, & Ellman J.A. (1994). The combinatorial synthesis and chemical and biological evaluation of a 1,4-benzodiazepine library.. Proceedings of the National Academy of Sciences of the United States of America, 91(11), 4708-4712.
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