Thymidine

Manufactured by Selleck Chemicals

Thymidine is a nucleoside that serves as a building block for DNA synthesis. It is an essential component for in vitro cell culture and molecular biology applications.

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Lab products found in correlation

Thymidine, by Merck Group (9 mentions) Nocodazole, by Merck Group (5 mentions) Ro 3306, by Selleck Chemicals (4 mentions) Rnaimax, by Thermo Fisher Scientific (4 mentions) Doxycycline, by Selleck Chemicals (2 mentions) Thymidine, by Bio-Techne (2 mentions) Doxycycline, by Merck Group (2 mentions) Zm447439, by Bio-Techne (2 mentions) Lonafarnib, by Selleck Chemicals (2 mentions) Mg132, by Merck Group (2 mentions) Ro 3306, by Bio-Techne (2 mentions) Hiperfect, by Qiagen (2 mentions) Mimosine, by Merck Group (2 mentions) Bi2536, by Selleck Chemicals (2 mentions) Nocodazole, by Selleck Chemicals (2 mentions) Cell observer, by Zeiss (1 mentions) Zen software, by Zeiss (1 mentions) Dulbecco s modified eagle s medium dmem, by Thermo Fisher Scientific (1 mentions) F12 medium, by Thermo Fisher Scientific (1 mentions) Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)

14 protocols using thymidine

Cell Cycle Synchronization and Perturbation

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To increase the number of mitotic cells, RPE1 cells and LCLs were treated with 3 mM or 2 mM thymidine (Sigma, T9250) for 22 h, respectively. The RPE1 cells were released for 8 h and the LCLs for 5 h before fixation. For synchronization at late G1 phase, RPE-1 cells were treated with 0.4 mM mimosine (Sigma, M0253) for 22 h. mimosine was washed out twice with fresh media. For synchronization in mitosis, RPE-1 cells were first treated with 3 mM thymidine for 22 h. After washing out thymidine, the cells were incubated with 5 μM paclitaxel (Selleck, S1150) for 12 h or 100 μM monastrol for 9 h. For inhibition of Plk1 activity, cells synchronized with paclitaxel were treated with 100 nM BI2536 (Selleck, S1109) for 3 h. For forced cell cycle exit, mitotic cells were treated with 2 μM ZM447439 (Selleck, S1103) for 1 h. In all, 0.5 mM IAA (Sigma, I5148) for CENP-E KO cells, 50 μM noscapine (Sigma, 363960), or 200 nM GSK923295 (APExBIO, A3450) for wild-type cells was added right after release from mimosine or thymidine.

Owa M, & Dynlacht B. (2021). A non-canonical function for Centromere-associated protein-E controls centrosome integrity and orientation of cell division. Communications Biology, 4, 358.

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Cell Cycle Synchronization Protocols

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Synchronization of cells at G1/S boundary was performed with a double thymidine block. Briefly, ~50% confluent cells were treated with 2 mM thymidine (Sigma-Aldrich) for 20 h, rinsed twice and released into drug-free media for 9 h, then treated again with 2 mM thymidine for 18 h. Synchronization of cells in prometaphase was done by addition of 100 ng/ml nocodazole (Selleckchem) 4 h after release from a single-thymidine block and incubated for 10 h. Mitotic cells were collected by mechanical shake off.

Cuevas-Navarro A., Van R., Cheng A., Urisman A., Castel P, & McCormick F. (2021). The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression. Current biology : CB, 31(17), 3915-3924.e9.

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Validating TANGO6 Gene Nuclear Localization

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To validate the genetic sequences of the NLS (Nuclear localization signal) in TANGO6 gene, we deleted the whole NLS by Seamless Cloning Kit (Beyotime) according to the manufacturer’s instruction. To perform site-directed mutagenesis, we precisely omitted or altered the sequence of targeted amino acids on the constructed pCS2-HA-TANGO6 plasmids following a standard protocol^{82 (link)}. In short, the primers were designed on both sides of the mutation site. The fragments were amplified by PCR and they were used to obtain the plasmids containing site-directed mutagenesis. To verify TM (transmembrane) and signal anchor domains, the original or modified elements were fused with the DsRed fragments by seamless cloning. For chemical treatment assay, the HeLa cells were treated with thapsigargin^{39 (link)} (2 μM, Selleck) for 24 h to induce ER stress. For topology detection and cell cycle synchronization (see below), digitonin (25 mg/mL, Sigma) or thymidine (2 μM, Selleck) were individually used to treat HeLa cells on ice for 5 min (digitonin) or for 16 h (thymidine).

Feng Z., Liu S., Su M., Song C., Lin C., Zhao F., Li Y., Zeng X., Zhu Y., Hou Y., Ren C., Zhang H., Yi P., Ji Y., Wang C., Li H., Ma M., Luo L, & Li L. (2024). TANGO6 regulates cell proliferation via COPI vesicle-mediated RPB2 nuclear entry. Nature Communications, 15, 2371.

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Cell Synchronization and Inhibitor Treatments

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HeLa, MDA-MB-231, U2OS, and 293 T cells were supplied with DMEM (Gibco) containing 10% fetal bovine serum (HyClone), 100 U/mL penicillin, and 100 mg/ml streptomycin (Gibco) in a 5% CO₂ humidified incubator at 37 °C. To synchronize cells in M phase, HeLa cells were blocked with 100 ng/mL Nocodazole (Sigma) for 16 h or treated with 2 mM Thymidine (Sigma) for 16 h and released to Thymidine-free medium for 10 h, followed by second exposure to 2 mM Thymidine for 16 h before being released for 9 h. The following reagents were also used: Taxol (1 µM, Selleck), LY294002 (50 µM, Selleck), and MK2206 (40 µM, Selleck).

Zhang X., Chen X., Liu J., Xu X., Zhang Y., Ruan Z., Xie Y., Huang Q., Yin T., Chen Z, & Chen S. (2017). Palladin is a novel microtubule-associated protein responsible for spindle orientation. Scientific Reports, 7, 11806.

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Visualizing Cell Cycle Regulation

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H2B-GFP-expressing control or KDM2A targeting sgRNA-transduced cells were seeded at 8 × 10⁴ cells per well in fibronectin-coated 12-well 1.5 mm glass-bottom wells (Cellvis). Following culture in DMEM medium supplemented with 15% FBS for 24 h, cells were synchronized in the G2 phase by sequential treatment of thymidine and CDK1 inhibitor Ro-3306 (S7747, Selleckchem). Cells were firstly cultured in a medium containing 2 mM thymidine (Sigma-Aldrich) for 24 h. After washing twice with PBS followed by once with growth media, the cells were then released into fresh medium for 2 h before treatment with 10 mM CDK1 inhibitor for 16 h. Finally, cells were washed twice with PBS and once with growth media before being subjected to live cell imaging with Zeiss Cell Observer (ZEISS). Cells were monitored for 8 h at 5 min intervals. Movies are output by Zeiss ZEN software (ZEISS).

Li F., Wang Y., Hwang I., Jang J.Y., Xu L., Deng Z., Yu E.Y., Cai Y., Wu C., Han Z., Huang Y.H., Huang X., Zhang L., Yao J., Lue N.F., Lieberman P.M., Ying H., Paik J, & Zheng H. (2023). Histone demethylase KDM2A is a selective vulnerability of cancers relying on alternative telomere maintenance. Nature Communications, 14, 1756.

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Breast Cancer Cell Line Culture

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MDA-MB-231, MDA-MB-468, HCC1806, HCC38, SUM159, T47D, MCF7, MCF10A, and IMR90 cell lines were purchased from American Type Culture Collection. HCC1937 and 293T cells were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Sciences. The above cells were cultured in DMEM, RPMI-1640, or F12 Medium with 10% fetal bovine serum in a humidified atmosphere containing 5% CO₂ at 37 °C. Dulbecco’s modified Eagle’s medium (DMEM), RPMI-1640 Medium, F12 Medium, and fetal bovine serum were purchased from Gibco, and Thymidine was purchased from Selleck.

Wang J., Dong Y., Ma H., Wu L., Zhen X., Tang L., Jin J., Han S., Zhang P, & Peng J. (2021). The deubiquitinase USP28 stabilizes the expression of RecQ family helicases and maintains the viability of triple negative breast cancer cells. The Journal of Biological Chemistry, 298(1), 101443.

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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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Cell Cycle Synchronization Protocol

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Cells were synchronized in the G2 phase by sequential treatment of thymidine and CDK1 inhibitor Ro-3306 (S7747, Selleckchem). Briefly, cells were first cultured in a medium containing 2 mM thymidine (Sigma-Aldrich) for 21 h. After washing twice with PBS followed by once with growth media, the cells were released into fresh medium for 3 h before treatment with 10 mM CDK1 inhibitor for 12 h.

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Synchronizing MCF7 Cells to Early S Phase

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To synchronize MCF7 cells to the early S phase, double thymidine block procedure was employed. Initially, when cells reached around 40% confluence, they were exposed to thymidine (2 mM, 15 h) (Selleck, Cat# S4803). Subsequently, they were released for 9 h and subjected to a second block with thymidine (2 mM, 15 h). After PBS washing extensively, the cells were exposed to nocodazole (0.1 μg/mL, 10 h) (Sigma, Cat# M1404). The arrested cells were washed with warm medium containing biotin-phenol (Adipogen, Cat# CDX-B0270) 3 times and released for 45 min to progress into the mitotic phase, after which they were utilized for the APEX labelling reaction.

Yue W., Li X., Zhan X., Wang L., Ma J., Bi M., Wang Q., Gu X., Xie B., Liu T., Guo H., Zhu X., Song C., Qiao J, & Li M. (2024). PARP inhibitors suppress tumours via centrosome error-induced senescence independent of DNA damage response. eBioMedicine, 103, 105129.

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Cell Cycle Synchronization Protocols

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For double thymidine block, PC-3 cells were treated with 2 mM thymidine (Selleck, catalog no. S4803) for 18 hours and were released into regular medium for 9 hours, after which the cells were further blocked by treatment with thymidine for another 15 hours. For thymidine-nocodazole block, PC-3 cells were treated with 2 mM thymidine for 24 hours and were released into regular medium for 3 hours, after which cells were further blocked by treatment with nocodazole (Selleck, catalog no. S2775) for another 12 hours. At the indicated time points after release, the cells were harvested for Western blot analysis using previously described methods.

Ma J., Ren D., Wang Z., Li W., Li L., Liu T., Ye Q., Lei Y., Jian Y., Ma B., Fan Y., Liu J., Gao Y., Jin X., Huang H, & Li L. (2024). CK2-dependent degradation of CBX3 dictates replication fork stalling and PARP inhibitor sensitivity. Science Advances, 10(21), eadk8908.

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