Polysomal profiling was done as described (Tcherkezian et al., 2014 (link); Culjkovic-Kraljacic et al., 2016 (link)). Briefly, cells were treated with cyclohexamide (100 µg/ml, Sigma Aldrich, Cat# C7698) 10 min before harvesting and lysates were prepared using polysome lysis buffer (15 mM Tris pH 7.4, 250 mM NaCl, 15 mM MgCl2, 1% Triton X-100, 100 g/ml cyclohexamide, 1 mM DTT, 400 U/ml RNase inhibitors and protease inhibitors (Sigma Aldrich, Cat# 11697498001). Equal amounts (10 mg) of protein lysates were layered on a 20–50% linear sucrose gradient (20% and 50% sucrose solutions in 15 mM Tris pH 7.4, 15 mM MgCl2, 150 mM NaCl, 1 mM DTT, 100 µg/Ml cyclohexamide and 20 U/ml RNase inhibitors), mixed on Gradient Station IP Biocomp and centrifuged in a Beckman SW41Ti rotor at 92,000 g for 3 hr at 4˚C. Following centrifugation, polysomal fractions were collected by continuously monitoring and recording the A254 on a Gradient Station IP (Biocomp) attached to a UV-MII (GE Healthcare, CA) spectrophotometer. RNAs were isolated from polysomal fractions using TRIzol reagent. RNAs from each fraction were monitored using RT-qPCR.
Cyclohexamide
Manufactured by Merck Group
Sourced in United States, Germany
Cyclohexamide is a laboratory reagent used in various scientific applications. It serves as an inhibitor of protein synthesis, specifically targeting the translocation step. Cyclohexamide is a widely used tool in cell biology research to study cellular processes and protein turnover.
Automatically generated - may contain errors
Lab products found in correlation
Mg132, by Merck Group (13 mentions)
Actinomycin d, by Merck Group (9 mentions)
Gradient station ip, by BioComp Instruments (5 mentions)
Uv mii, by GE Healthcare (5 mentions)
Rapamycin, by Merck Group (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Triton x 100, by Avantor (4 mentions)
Rnase inhibitors, by BioComp Instruments (3 mentions)
Protease inhibitor, by Merck Group (3 mentions)
Sw41ti rotor, by Beckman Coulter (3 mentions)
Rnasin, by Promega (3 mentions)
Anti β actin, by Merck Group (3 mentions)
Mg132, by Selleck Chemicals (3 mentions)
Vancomycin, by Merck Group (3 mentions)
Cycloheximide (chx), by Merck Group (3 mentions)
Trimethoprim, by Merck Group (3 mentions)
Etoposide, by Merck Group (3 mentions)
Chloroquine, by Merck Group (3 mentions)
Rnase inhibitor, by Thermo Fisher Scientific (3 mentions)
Withaferin a, by Merck Group (2 mentions)
114 protocols using cyclohexamide
1
Polysomal Profiling for Translational Regulation
2
Polysome Fractionation Workflow
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Stocks for polysome buffer (PSB; 50 mM Tris, pH = 7.5, 100 mM KCl, 12 mM MgCl2, 1% IPEGAL CA-630 [Sigma-Aldrich], plus: 1 mM DTT, 60 U/ml RiboLock RNase inhibitor [Thermo Fisher Scientific], 100 μg/ml Cyclohexamide [Sigma-Aldrich], 2× SigmaFast EDTA-free protease inhibitor cocktail [Sigma-Aldrich] dissolved in PSB stock), high salt wash buffer (HSB; 50 mM Tris pH = 7.5, 300 mM KCl, 12 mM MgCl2, 1% IPEGAL, plus: 1 mM DTT, 20 U/ml RiboLock, 100 μg/ml Cyclohexamide and 0.5× EDTA-free SigmaFast protease inhibitor cocktail), and extra high salt buffer (EHSB; HSB containing additional 300 mM NaCl) were prepared using RNase-free reagents and stored at 4°C. Inhibitors were added to stock solutions directly before use (indicated by “plus”). RNA purifications were performed in a Biosafety level 3 environment. Frozen tissue samples were homogenized at 450 rpm, using Wheaton Potter-Elvehjem homogenizers and PTFE pestles (DWK Life Science) with a motorized homogenizer (HEI-Torque Core, heidolph), in 200 μl ice-cold PSB per 0.01 g tissue. Homogenates were centrifuged at 4°C, 400g for 2 min to collect nuclei. Supernatant was transferred to fresh vials and centrifuged at 4°C, 10,000g for 10 min.
3
Ribosome profiling of organoids
4
Minigene Transfection and Splicing Analysis
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Minigene plasmids (0.2 μg) were transfected according to the Lipofectamine 3000 protocol (Invitrogen, Cat #L30000015) and transfected into HEK293T cells in DMEM supplemented with 10% FBS for 24 h before RNA isolation with Trizol (Fisher Scientific Company LLC, Cat #15596018). P1 cells were treated with 100 mg/mL cyclohexamide (Sigma Aldrich, Cat #C0934) for 2 h before collection.
PMO treatments were carried out in 24 well plates. HEK293T cells (1.1 × 105) were seeded and transfected the next day with the minigene DNA transfection mix and Lipofectamine 3000. The minigene transfection mix was incubated for 6 h and then the oligomer solutions were added to each well in DMEM supplemented with 10% FBS at a final concentration of 10 μM, and the plate was gently swirled after each oligomer addition. Endo-Porter (8 μM, Gene Tools, SKU: OT-EP-PEG1) was added directly to each well and the plate was vigorously swirled after each addition. Cells were incubated 24 h before RNA isolation, harvested using Trizol, and splicing evaluation was performed by PCR analysis described below. P1 cells were treated with 100 mg/mL cyclohexamide (Sigma Aldrich, Cat #C0934) for 2 h before collection.
PMO treatments were carried out in 24 well plates. HEK293T cells (1.1 × 105) were seeded and transfected the next day with the minigene DNA transfection mix and Lipofectamine 3000. The minigene transfection mix was incubated for 6 h and then the oligomer solutions were added to each well in DMEM supplemented with 10% FBS at a final concentration of 10 μM, and the plate was gently swirled after each oligomer addition. Endo-Porter (8 μM, Gene Tools, SKU: OT-EP-PEG1) was added directly to each well and the plate was vigorously swirled after each addition. Cells were incubated 24 h before RNA isolation, harvested using Trizol, and splicing evaluation was performed by PCR analysis described below. P1 cells were treated with 100 mg/mL cyclohexamide (Sigma Aldrich, Cat #C0934) for 2 h before collection.
5
PINK1 Submitochondrial Localization Assay
6
AML Cell Line Authentication and Inhibitor Use
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Human AML cell lines were purchased from the ATCC (THP1) and DSMZ (OCI-AML3, U937, MV4;11 and SHI-1), authenticated by short tandem repeat profiling using the PowerPlex 16 system (Promega, Southampton, UK) and mycoplasma negative status confirmed using the MycoAlert Mycoplasma Detection Kit (Lonza, Verviers, Belgium). The following reagents and inhibitors were used, Withaferin A (Merck Life Science UK, Dorset, UK and Cayman Chemical, Ann Arbor, MI, United States); Cyclohexamide, Thapsigargin and MG132 (Merck Life Science UK); Actinomycin D (Cambridge Bioscience, Cambridge, UK); pifithrin-μ. (Abcam, Cambridge, UK).
7
Cytokine Signaling Inhibition Assay
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BMMCs (106 cells/ml) were IL-3-starved (1 h), pre-incubated (30 min) with SP600125 (JNK-inhibitor), SU6656 (SFK-inhibitor), IKK-inhibitors (VII, PS-1145, BMS-34554) and U73122 (PLC inhibitor) (If not other stated all these inhibitors were used in a concentration of 5 μM). Furthermore we used the protein biosynthesis inhibitor cyclohexamide (340 μM), the PLD1 inhibitor CAY10594 (10 μM), and the Ca2+ chelator BAPTA-AM (10 μM) or ionomycin (in a concentration from 0,5–10 ng/ml) (all inhibitors were from Merck Millipore). After pre-incubation of the indicated inhibitor cells were stimulated with IL-3 and/or IL-33 (Peprotec., in a concentration of 50 ng/ml). V2D1 cells were seeded (106 cells/ml), pre-treated with the IKK-inhibitor VII (1 h). Cells were lysed (in 20 mM HEPES, pH7.5; 10 mM EGTA; 40 mM β-glycerophosphate; 2.5 mM MgCl2; 2 mM orthovanadate; 1 mM dithiothreitol; 20 μg/ml aprotinin; 20 μg/ml leupeptin, 1% Triton). Protein concentration was determined by using the BCA-assay (Pierce) and samples were boiled in 6 x Laemmli buffer.
8
Activating Mammalian Zygote Development
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Nocodazole (Sigma-Aldrich #M1404) was diluted freshly in hybridoma grade DMSO (Sigma-Aldrich #D2650) to make a 20 mM stock and was added to zygotes at the final concentration of 20 μM, at least 9 hours before NEBD. Ionomycin (Sigma-Aldrich #I0634) was diluted in hybridoma grade DMSO (Sigma-Aldrich D2650) to make a 1.35 mM stock (1 mg/ml). The stock was stored at −20°C for no longer than 3 months. Ionomycin was added to eggs at the final concentration of 5 μM (3.7 μg/ml) for 5 minutes. Cytochalasin B (Sigma-Aldrich #C6762) was diluted in hybridoma grade DMSO (Sigma-Aldrich D2650) to make a 10 mg/ml stock and was added to activated eggs at the final concentation of 5 μg/ml. Cyclohexamide (Merck #239763) was diluted in embryo tested water (Sigma-Aldrich #TMS-006-C) to make a 10 mg/ml stock and was added to activated eggs at a final concentation of 10 μg/ml. Heparin (Merck Millipore #375095-100KU) was diluted in embryo tested water (Sigma-Aldrich #TMS-006-C) to make a 5000 IU/ml stock.
9
AML Cell Line Authentication and Inhibitor Use
10
Comprehensive Analysis of Bioactive Compounds
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Jasmonic acid, yeast extract, ferrozine (3-(2-pyridyl)-5,6-bis-(4-phenyl-sulfonic acid)-1,2,4-triazine), 2,20-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), α-amylase, pancreatin, pepsin, lipase, α-glucosidase, bile extract, lipoxygenase, linoleic acid, angiotensin converting enzyme, o-phthalaldehyde, p-nitrophenyl acetate, dimethyl sulfoxide, 3,5-dinitrosalicylic acid, p-nitrophenol, protocatechuic acid, syringic acid, vanillic acid, sinapic acid, salicylic acid, caffeic acid, and hydroxybenzoic acid, cyclohexamide, resazurin were purchased from Sigma-Aldrich (Poznan, Poland). The COX Activity Assay kit was purchased from Cayman Chemical company (Ann Arbor, MI, USA). Penicillin and streptomycin were purchased from Life Technologies, Warsaw, Poland. Mueller–Hinton broth, and Mueller–Hinton agar were also obtained (Biomaxima, Lublin, Poland). All other chemicals were of analytical grade.
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