Uridine

Manufactured by Merck Group

Sourced in United States, Germany, China, United Kingdom, France, Japan, Sao Tome and Principe

Uridine is a nucleoside that is a constituent of ribonucleic acid (RNA). It serves as a precursor in the biosynthesis of uridine triphosphate (UTP) and other pyrimidine nucleotides. Uridine can be used as a research tool in various biological and biochemical applications.

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Lab products found in correlation

351 protocols using uridine

SLAM-seq analysis of mRNA dynamics

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SLAM-seq was performed as described previously (Herzog et al., 2017 (link)) with minor modifications. HEK293T (WT and PCIF1 KO) cells (at 60% confluency) were incubated with cell culture growth medium supplemented with 25 μM 4-thiouridine (s⁴U) for 24 h (pulse phase). s⁴U incorporation was confirmed by HPLC analysis, as previously described (Herzog et al., 2017 (link)). The uridine chase was initiated by changing media containing 2.5 mM uridine (Sigma) and cells were collected for RNA extraction after 6 and 12 h. The 0 h sample were the cells that have completed the pulse with s⁴U, but without uridine-chase. Total RNA was extracted using RNAzol reagent (MRC) according to the manufacturer’s instructions, maintaining reducing conditions to prevent oxidation of s⁴U (0.1 mM DTT final concentration). For thiol alkylation, a master mix (10 mM iodoacetamide, 50 mM NaPO₄ pH 8 and 50% DMSO) was prepared, centrifuged, and added to 20 μg of total RNA at 50°C for 15 min and then purified by ethanol precipitation. After that, two rounds of poly(A) mRNA enrichment was carried out with oligo d(T)25 Magnetic Beads (NEB). Standard RNA-seq libraries were prepared using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) following the instructions of the manufacturer. Sequencing was performed on a HiSeq2500 (Illumina) with 50 nucleotide reads.

Boulias K., Toczydłowska-Socha D., Hawley B.R., Liberman N., Takashima K., Zaccara S., Guez T., Vasseur J.J., Debart F., Aravind L., Jaffrey S.R, & Greer E.L. (2019). Identification of the m6Am methyltransferase PCIF1 reveals the location and functions of m6Am in the transcriptome. Molecular cell, 75(3), 631-643.e8.

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Uridine Treatment of Premature Aging MSCs

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For hMSC uridine treatment, during the entire experiments, WS hMSCs (passage, P5), HGPS hMSCs (P10) and hPMSCs (P13) of the uridine treatment group were cultured in hMSC culture medium supplemented with 200, 100 and 100 μM uridine (Sigma, U3003), respectively. The cells of the vehicle group were cultured in hMSC culture medium. Each group of cells with three biological replicates were seeded into 6-well plates (30,000 cells for HGPS and WS hMSCs and 15,000 cells for hPMSCs per well) and cultured. Immunostaining of Ki67, H3K9me3, γH2A.X and 53BP1, cell cycle assay and RT-qPCR analysis of repetitive element transcripts in vehicle- or uridine-treated WS hMSCs, HGPS hMSCs and hPMSCs were conducted after two passages with vehicle or uridine treatment.

Liu Z., Li W., Geng L., Sun L., Wang Q., Yu Y., Yan P., Liang C., Ren J., Song M., Zhao Q., Lei J., Cai Y., Li J., Yan K., Wu Z., Chu Q., Li J., Wang S., Li C., Han J.D., Hernandez-Benitez R., Shyh-Chang N., Belmonte J.C., Zhang W., Qu J, & Liu G.H. (2022). Cross-species metabolomic analysis identifies uridine as a potent regeneration promoting factor. Cell Discovery, 8, 6.

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Establishment of ρ0 Cancer Cell Lines

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Human cancer cell lines HeLa and SAS were obtained from the Cell Resource Center for Biomedical Research, Institute of Development, Aging and Cancer, Tohoku University (Sendai, Japan). ρ⁰ cells were established through culture in RPMI1640 (Wako Pure Chemical Industries Ltd., Osaka, Japan) containing 5% FBS (Gibco Invitrogen Corp., Carlsbad, CA, USA), 50 ng/mL ethidium bromide (Nacalai Tesque Inc., Kyoto, Japan), 50 μg/mL uridine (Sigma‐Aldrich, St Louis, MO, USA), and 110 μg/mL sodium pyruvate (Sigma‐Aldrich) for 3‐4 wk.17 Cells were maintained in RPMI 1640 supplemented with 10% FBS, 50 μg/mL uridine, and 110 μg/mL sodium pyruvate in a humidified atmosphere at 37°C with 5% carbon dioxide. Exponentially growing cells were used in all experiments.

Tomita K., Takashi Y., Ouchi Y., Kuwahara Y., Igarashi K., Nagasawa T., Nabika H., Kurimasa A., Fukumoto M., Nishitani Y, & Sato T. (2019). Lipid peroxidation increases hydrogen peroxide permeability leading to cell death in cancer cell lines that lack mtDNA. Cancer Science, 110(9), 2856-2866.

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Modulating Mutant HTT Expression

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The mouse striatal neuron cell lines, ST Hdh^Q7/Q7 and ST Hdh^Q111/Q111, were incubated with various concentrations of 6-AZA for 12 h, and total RNA was collected for analysis of gene expression. Analogously, cells treated with 6-AZA for 24 h were collected for Western blot analysis. To assess cell viability, adherent cells were collected from plates at the end of treatment and the number of viable cells were counted using hemocytometer after Trypan blue staining. In uridine supplementation experiments, ST Hdh^Q111/Q111 cells were seeded at a density of 8 × 10⁵ cells/well in 6-well plates and treated with 6-AZA (10 µM) plus indicated concentrations of uridine (Sigma) for 24 h. The cells were subjected to assess the number of viable cells by Trypan blue exclusion assay and determine the expression of mutant HTT by Western blot assay.

Deng N., Wu Y.Y., Feng Y., Hsieh W.C., Song J.S., Lin Y.S., Tseng Y.H., Liao W.J., Chu Y.F., Liu Y.C., Chang E.C., Liu C.R., Sheu S.Y., Su M.T., Kuo H.C., Cohen S.N, & Cheng T.H. (2022). Chemical interference with DSIF complex formation lowers synthesis of mutant huntingtin gene products and curtails mutant phenotypes. Proceedings of the National Academy of Sciences of the United States of America, 119(32), e2204779119.

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SLAM-seq protocol for mESC analysis

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See Protocol Exchange for detailed information regarding SLAM-seq37 (link). mESCs were seeded the day before the experiment at a density of 10⁵ cells/ml. s⁴U-metabolic labeling in mESCs was performed by incubating mESCs in standard medium but adding s⁴U (Sigma) to a final concentration of 100 µM and media exchange every 3 hours for the duration of the pulse. For the uridine chase experiment, cells were washed twice with 1x PBS and incubated with standard medium supplemented with 10 mM uridine (Sigma). At respective time points, cells were harvested followed by total RNA extraction using TRIzol (Ambion) following the manufacturer’s instructions but including 0.1mM DTT (final conc.) during isopropanol precipitation. RNA was resuspended in 1 mM DTT. For a typical SLAM-seq experiment, 5 µg total RNA were treated with 10 mM iodoacetamide under optimal reaction conditions and subsequently ethanol precipitated and subjected to Quant-seq 3′ end mRNA library preparation.

Herzog V.A., Reichholf B., Neumann T., Rescheneder P., Bhat P., Burkard T.R., Wlotzka W., von Haeseler A., Zuber J, & Ameres S.L. (2017). Thiol-linked alkylation of RNA to assess expression dynamics. Nature methods, 14(12), 1198-1204.

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Pulse-Chase Assay for Stem Cell States

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mESCs were seeded at a density of 3×10⁵ cells/mL the day before the experiment. 4sU metabolic labeling was performed by incubating mESCs in fresh medium supplemented with 200 μM 4sU and media exchange every 4 hours for the duration of the 24-hour pulse. For the uridine chase experiment, cells were washed twice with PBS and incubated with fresh medium supplemented with 10 mM uridine (Sigma, U6381). At respective time points (0/0.5/1/3/6/12/24-hour), cells were harvested, methanol fixed as aforementioned and stored at -80°C for later use. On the day of performing droplet microfluidics assays, all samples were rehydrated (PBS-based buffer) and analyzed in parallel. Clustering analysis based on total RNAs separated mESCs (combined from 7 time points) into three stem cell states (pluripotent: 97.4%+/-0.78%, intermediate: 1.5%+/-0.49%, and 2C-like: 1.1%+/-0.38%), suggesting that our pulse-chase experiments did not significantly alter the state transition.

Qiu Q., Hu P., Qiu X., Govek K.W., Camara P.G, & Wu H. (2020). Massively parallel and time-resolved RNA sequencing in single cells with scNT-Seq. Nature methods, 17(10), 991-1001.

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Thermoregulation Effects of Uridine and Glucose

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Male WT (15 to 35 weeks old) and age-matched ob/ob mice were used for body temperature measurements through an IPTT-300 transponder implanted longitudinally above the shoulder of mouse (Bio Medic Data Systems). To study the thermal effect of uridine, 0.1 g/ml uridine (Sigma) in PBS was administrated to mice (1 g/kg body weight) via intraperitoneal injection or oral gavage. To study the thermal effect of glucose, 0.25 g/ml glucose (Sigma) in H₂O was administrated orally to mice (2.5 g/kg body weight). To study the combined effect of glucose and uridine, glucose-uridine solution (0.25 g/ml glucose and 0.1 g/ml uridine in H₂O) was administrated orally at the same dose as the study for glucose alone. All the treatment and temperature measurements were performed at ambient room temperature (23° to 25°C), if not specified. A refrigerated incubator at 29°C (Powers Scientific) was used to achieve a near-thermoneutral environment.

Deng Y., Wang Z.V., Gordillo R., An Y., Zhang C., Liang Q., Yoshino J., Cautivo K.M., De Brabander J., Elmquist J.K., Horton J.D., Hill J.A., Klein S, & Scherer P.E. (2017). An adipo-biliary-uridine axis that regulates energy homeostasis. Science (New York, N.Y.), 355(6330), eaaf5375.

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SDHB Knockout in HCT-116 Cells

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HCT-116 cells were plated at 15000 cells/3 mL/well on 6-well plates (Corning) and cultured overnight. The next day, the cells were transfected with 1 μg of SDHB clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 knockout plasmid (Santa Cruz) and SDHB homology directed repair (HDR) plasmid (Santa Cruz) or a control CRISPR/Cas9 plasmid (Santa Cruz) using Lipofectamine 2000 (Thermo Fisher Scientific) as described by the manufacturer. After forty-eight hours post transfection, cells were selected with 2 μg/μL of puromycin (Sigma) for 1 week. Cells transfected with the SDHB knockout plasmid were collected as SDHB knockout polyclonal cells. Cells were cloned by limited dilution on 96-well plates (Corning), and cloned cells were collected for analysis of protein expression and genome copy number of SDHB. After transfection of SDHB knockout plasmid, 1 mM sodium pyruvate (Gibco) and 50 μg/ml of uridine (Sigma) were added to the culture medium described above because cells that lack a functional mitochondrial electron transport chain such as ρ0 cells fail to proliferate without supra-physiological levels of uridine and pyruvate in the culture medium [28 (link)].

Kitazawa S., Ebara S., Ando A., Baba Y., Satomi Y., Soga T, & Hara T. (2017). Succinate dehydrogenase B-deficient cancer cells are highly sensitive to bromodomain and extra-terminal inhibitors. Oncotarget, 8(17), 28922-28938.

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Evaluating Cytotoxicity of Bioactive Compounds

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HepG2 cells (5 × 103 cells/96-well plate) were incubated for 24 hours, and starvation was performed for 4 hours. Uridine, N-acetylmannosamine, cinobufagin, and cinnamoylglycine were purchased from Sigma (St. Louis, MO, USA) and dissolved in dimethyl sulfoxide (DMSO). HepG2 cells were treated with different concentrations of Uridine, N-acetylmannosamine, cinobufagin, and cinnamoylglycine for 24 hours, and then incubated with 3-(4, 5-dimethyl-2-thiazolyl)−2, 5-diphenyltetrazolium bromide (MTT, Sigma Aldrich) for 4 hours at 37°C and 5% Co₂ incubator. The formazan crystals were dissolved in DMSO and their absorbance was measured at 570 nm using a microplate reader (Molecular Devices, Silicon Valley, CA, USA).

Park Y.R., Lee H.L., Hyun J.Y., Choi J., Moon J.H., Kim B.Y., Yang S.J., Lee J.H., Kim B.K., Park T.S., Suk K.T, & Lee D.Y. (2023). Systemic multiomics evaluation of the therapeutic effect of Bacteroides species on liver cirrhosis in male mice. Microbiology Spectrum, 11(6), e05349-22.

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Measuring RNA Stability by BrU Pulse-Chase

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RNA half-life measurements by BrU pulse-chase was carried out essentially as described previously^{44 (link)}. Briefly, HEK293T cells were pulsed with 150 μM 5-bromouridine (Santa Cruz Biotechnology) for 24 h. Chase was initiated by changing to medium containing 1.5 mM uridine (Sigma) and cells were collected for RNA extraction after 6 and 16 h. BrU-pulsed cells without uridine-chase were used as basal (0 h) controls. Total RNA was extracted with TRIzol reagent (Thermo Fisher Scientific) according to the manufacturers instructions. Immunoprecipitation of BrU-labelled RNA from total RNA was carried out as previously described^{44 (link)}. A BrU-labelled NanoLuc luciferase (nLuc) RNA was generated by in vitro transcription as previously described^{44 (link)} and used as a spike-in immunoprecipitation control at 10 pgp per 1 μg input RNA.

Mauer J., Luo X., Blanjoie A., Jiao X., Grozhik A.V., Patil D.P., Linder B., Pickering B.F., Vasseur J.J., Chen Q., Gross S.S., Elemento O., Debart F., Kiledjian M, & Jaffrey S.R. (2016). Reversible methylation of m6Am in the 5′ cap controls mRNA stability. Nature, 541(7637), 371-375.

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