Thymidine

Manufactured by Bio-Techne

Thymidine is a nucleoside that is a naturally occurring component of DNA. It functions as a building block for DNA synthesis and plays a critical role in cellular replication processes.

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Lab products found in correlation

Ro 3306, by Bio-Techne (3 mentions) Thymidine, by Merck Group (3 mentions) Zm447439, by Bio-Techne (3 mentions) Rnaimax, by Thermo Fisher Scientific (2 mentions) Mg132, by Merck Group (2 mentions) Doxycycline, by Merck Group (2 mentions) Nocodazole, by Merck Group (2 mentions) Doxycycline, by Selleck Chemicals (2 mentions) Thymidine, by Selleck Chemicals (2 mentions) Lonafarnib, by Selleck Chemicals (2 mentions) Hiperfect, by Qiagen (2 mentions) Thymidine, by Thermo Fisher Scientific (1 mentions) Hy 18682, by MedChemExpress (1 mentions) Advanced dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions) Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions) Cos 7, by American Type Culture Collection (1 mentions)

5 protocols using thymidine

Cell Cycle Arrest and Centrosome Inhibition in Chlamydia Infection

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Mock or Chlamydia-infected RPE-1 cells were arrested in S-phase by incubating them with 2mM thymidine (ACROS Organics, 226740050) in standard growth medium for 24 hours, starting at 12 hpi. Cells were arrested in G2 by first incubating them with 2mM thymidine for 18 hours, followed by release from the thymidine block for 6 hours and incubation with 10μM RO-3306 (TOCRIS, cat # 4181) in standard medium for 12 hours. Both the S-phase and the G2 arrest experiments were analyzed at 36 hpi using immunofluorescence microscopy.
To inhibit centrosome duplication, Chlamydia, HPV and HPV+Chlamydia cells were incubated with 125nM of the Plk4 inhibitor centrinone (MedChemExpress HY-18682) in standard growth medium starting at 1 hpi. The experiment was evaluated by immunofluorescence microscopy at 36 hpi.

Wang K., Muñoz K.J., Tan M, & Sütterlin C. (2021). Chlamydia and HPV induce centrosome amplification in the host cell through additive mechanisms. Cellular microbiology.

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Cell Cycle Synchronization and Analysis

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RPE-1 cells were treated with 2mM thymidine (Tocris, Minneapolis) for 17hrs, at which point the cells were washed with PBS and put in fresh media to allow for recovery. After 6 hours in fresh medium, the cells were treated with 10uM RO-3306 (Acros Organics, New Jersey) or 12hrs, and then washed and transferred into fresh media. At the indicated time points post-release, cells were trypsinized and collected into FACs tubes. Cells were pelleted at 300g for 5 minutes at 4°C, then resuspended in 2mL of cold PBS and spun at 300g for 5 minutes at 4°C. Pellets were then fixed in ice cold 70% ethanol, which was added dropwise while vortexing. Resuspended pellets were then stored for further fixation at -20°C for 24hrs. Fixed cells were pelleted at 300g for 5 minutes at 4°C, then resuspended in 20ug/mL propidium iodide and 0.125mg/mL RNase A in cold PBS for 2–4 hours at 4°C. Flow cytometry analysis was performed on the Attune NxT flow cytometer. FlowJo was used to generate the graphs. Singlet nuclei were gated based on scatter properties; 1N and 2N peaks were identified via the YL2-A channel.

Tormanen K., Ton C., Waring B.M., Wang K, & Sütterlin C. (2019). Function of Golgi-centrosome proximity in RPE-1 cells. PLoS ONE, 14(4), e0215215.

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Efficient Knockdown and Rescue in Cell Cycle

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For knockdown experiments, siRNAs (see Supplementary Table 1 for sequences and concentrations) were transfected using Hiperfect (Qiagen) or RNAi Max (Thermo Fisher Scientific) according to manufacturer’s instructions. After 16 hours of siRNA treatment, cells were arrested in S-phase by addition of thymidine (2 mM; Sigma-Aldrich Cat#T1895). For the rescue experiments, doxycycline (1 μg/ml; Sigma-Aldrich Cat#D9891) was also added at this point to induce the expression of the constructs. In the experiments in which farnesyl transferase activity was inhibited, Lonafarnib (5 μM; Selleckchem Cat#: S2797) was added together with thymidine, and the induction of the constructs with doxycycline was performed 8 hours later. After 24 hours of thymidine addition, cells were released and treated with the indicated drugs [ZM-447439 (2 μM; Tocris Bioscience, Cat#2458); BI-2536 (100 nM; Advanced ChemBlocks Cat#10293); Cpd-5 (250 nM; gift from R.H. Medema); RO-3306 (10 μM; Tocris Bioscience Cat#4181); Nocodazole (3.3 μM; Sigma-Aldrich Cat#M1404); MG-132 (5 mM), Cat#C2211]. Cells were used for experiments between 6-10 hours after thymidine release.

Sacristan C., Ahmad M.U., Keller J., Fermie J., Groenewold V., Tromer E., Fish A., Melero R., Carazo J.M., Klumperman J., Musacchio A., Perrakis A, & Kops G.J. (2018). Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis. Nature cell biology, 20(7), 800-810.

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HeLa and COS-7 Cell Cycle Synchronization

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HeLa and COS-7 cells from the American Type Culture Collection were maintained in advanced Dulbecco’s modified Eagle’s medium (Gibco) with 10% fetal bovine serum (FBS; Hyclone) and penicillin–streptomycin (100 units/ml and 100 μg/ml, respectively; Gibco) at 37°C with 8% CO₂.
For cell cycle synchronization, HeLa cells were first blocked in G1/S with 2.5 mM thymidine (Sigma) for 16 h and then released in fresh culture medium for 8 h to enrich mitotic cells. To inhibit Aurora B kinase activity, cells were treated with the Aurora B inhibitor ZM447439 (2458, TOCRIS) at 5 μM for 45 min after thymidine release.

Zhang M., Yang F., Wang W., Zohbi N., Wang X., Wang D., Zhuang X., Dou Z., Liu D., Song X., Green H.N., Liu X, & Yao X. (2021). SKAP interacts with Aurora B to guide end-on capture of spindle microtubules via phase separation. Journal of Molecular Cell Biology, 13(12), 841-852.

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Efficient Knockdown and Rescue in Cell Cycle

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