Atmi ku 55933

Manufactured by Selleck Chemicals

ATMi KU-55933 is a laboratory equipment product that functions as an inhibitor. It is designed to inhibit the activity of the ATM (Ataxia Telangiectasia Mutated) protein kinase.

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Ku-55933 (Ku) (2 citations)

7 protocols using atmi ku 55933

Maintaining and Characterizing ALT and Telomere-related Cell Lines

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All cell lines were grown at 37 °C under standard cell culture conditions (humidified atmosphere, 5% CO₂). ALT-positive GM847, SW26, and U-2 OS cells, as well as derived cell lines, and ALT-negative HeLa, HeLa LT (long telomeres), SW39 and hTERT-RPE1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (GIBCO) and penicillin-streptomycin antibiotics. For U-2 OS CycE/RAD52 WT and KO cells G418 400 μg/ml (Gibco, 10131-027), puromycin 1 μg/ml (Sigma, P8833) and tetracycline 2 μg/ml (Sigma, T7660) were added to the medium. U-2 OS 53BP1-GFP/RPA70-mScarlet cells were maintained in presence of puromycin 0.5 μg/ml (Sigma, P8833) and 5 μg/ml blasticidin (InvivoGen, ant-bl-05). All cells used in this study were grown under sterile conditions and routinely (monthly) tested for mycoplasma contamination and scored negative. The following compounds were used in this manuscript at the indicated final concentrations unless stated otherwise: ATRi Az-20 (5 μM for RPE-1 cells, else 1 μM, Tocris), ATRi VE-821 (5 μM, Selleckchem), APH (0,2 μM, Sigma-Aldrich), HU (2 mM, Sigma-Aldrich), Nocodazole (50 ng/ml, Sigma-Aldrich), CDKi RO-3306 (9 μM, Sigma-Aldrich), Colcemid (0,1 μg/ml, Roche), ATMi KU-55933 (10 μM, Selleckchem).

Lezaja A., Panagopoulos A., Wen Y., Carvalho E., Imhof R, & Altmeyer M. (2021). RPA shields inherited DNA lesions for post-mitotic DNA synthesis. Nature Communications, 12, 3827.

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Hematopoietic Stem Cell Culture Protocol

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Cells were clone sorted into 96-well round-bottom plates. HSCs were cultured in S-Clone (Iwai North America Inc.) supplemented with 0.75% AlbuMAX-I (Gibco), 1x penicillin/streptomycin, 50 mM 2-mercaptoethanol (Invitrogen) and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-12. Myeloid progenitors and c-kit enriched cells were cultured in Dulbecco’s modified Eagle’s medium and F-12 medium (Gibco and Invitrogen) supplemented with 10% fetal calf serum (Hyclone and Thermo Scientific), 1x penicillin/streptomycin, 2 mM GlutaMAX, 50 mM 2-mercaptoethanol (Invitrogen), and the following cytokines: 20 ng/ml mouse stem cell factor, 20 ng/ml mouse thrombopoietin, 20 ng/ml mouse IL-3, 20 ng/ml mouse granulocyte macrophage colony-stimulating factor (all purchased from PeproTech). c-kit enriched bone marrow (2×10⁶ cells/ml) were exposed for 4 hours to 10 μM ATMi (KU55933, Selleckchem). All cells were kept in a 5% O₂ incubator.

Gutierrez-Martinez P., Hogdal L., Nagai M., Kruta M., Singh R., Sarosiek K., Nussenzweig A., Beerman I., Letai A, & Rossi D.J. (2018). Diminished apoptotic priming and ATM signalling confer a survival advantage onto aged haematopoietic stem cells in response to DNA damage. Nature cell biology, 20(4), 413-421.

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Generating stable HeLa cell line for DNA damage response

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Cells were grown in DMEM (Life Technologies, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) at 37ºC/5% CO₂. The stable HeLa cell line (mCherry-53BP1-FFR) used in Figure 1 was generated by transfecting a plasmid containing mCherry-53BP1_1220-1711 (Addgene, Cambridge, MA; Dimitrova et al., 2008 (link)) into a cell line stably expressing GFP-tubulin (Mackay et al., 2010a (link)) using Lipofectamine LTX (Life Technologies) and selecting with 0.5 mg/ml G418 plus 0.5 μg/ml puromycin. Generation of the HeLa cell line stably expressing GFP-tubulin and histone H2B-mCherry was previously described (Mackay et al., 2010a (link)).
To induce replication stress, cells were treated for 24 h with either 50 μM hydroxyurea (MP Biomedicals, Santa Ana, CA) or 0.4 μM aphidicolin (Fisher Bioreagents). Where indicated, inhibitors were used at the following concentrations: AurBi (ZM447439; Biotechne, Minneapolis, MN), 2 μM; Chk1i (AZD7762; Selleck Chemicals, Houston, TX), 2 μM; ATRi (NU6027; EMD Millipore, Billerica, MA), 10 μM; ATMi (KU55933; Selleck Chemicals), 10 μM; and Chk2i (Chk2 inhibitor II; Millipore), 10 μM. As a control for the identification of DNA ultrafine bridges, ICRF-159 (Sigma-Aldrich, St. Louis, MO) was used at a concentration of 10 μM (Chan et al., 2007 (link)).

Mackay D.R, & Ullman K.S. (2015). ATR and a Chk1-Aurora B pathway coordinate postmitotic genome surveillance with cytokinetic abscission. Molecular Biology of the Cell, 26(12), 2217-2226.

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Maintenance and Treatment of Human Cell Lines

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Human cervical cancer HeLa cells, human embryonic kidney HEK293T cells, human osteosarcoma U2OS cells, human lung cancer A549 cells, human colon cancer HCT116 cells and LoVo cells were obtained from ATCC (American Type Culture Collection). DR-U2OS and EJ5-U2OS cells were gifts from Dr. Xingzhi Xu (Shenzhen University, China). These cells were maintained in McCoy’s 5A (M&C, 10051) medium or DMEM (M&C, 15019) supplemented with 10% FBS (Hyclone, SV30087.02) in a humidified incubator containing 5% CO₂. AO3_1 cells were provided by Dr. Qinong Ye (Beijing Institute of Biotechnology, China) and maintained in Ham's F12 (M&C, 10081) medium. Etoposide (VP16) (Sigma, E1383) and doxorubicin (DOX) (Sigma, D1515) were stored in DMSO at 80 mM and 10 mM stock concentrations, respectively. Cisplatin was purchased from TRC and ATMi (KU55933) was purchased from Selleck.

Hou T., Cao Z., Zhang J., Tang M., Tian Y., Li Y., Lu X., Chen Y., Wang H., Wei F.Z., Wang L., Yang Y., Zhao Y., Wang Z., Wang H, & Zhu W.G. (2020). SIRT6 coordinates with CHD4 to promote chromatin relaxation and DNA repair. Nucleic Acids Research, 48(6), 2982-3000.

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DNA Damage Induction in Cancer Cell Lines

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Colorectal cancer (CRC) cell lines (HCT116, HT29, WIDR and T87), lung cancer cell lines (A549, H460, H1299 and H1975), a human bronchial epithelial cell line (BEAS-2B), human glioma cell lines (U251, U373 and U87), human hepatoma cell lines (HuH7, 7721, HepG2) and human embryonic kidney 293 cells (HEK 293T) were all purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China). The culture conditions used in this study are shown in Supplementary Table S1. High glucose Dulbecco’s modified Eagle’s medium (DMEM) and RPMI-1640 medium were supplied by HyClone (Thermo Fisher, USA). All culture media were supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher), 100 U/mL penicillin and 100 µg/mL phytomycin (Gibco, Thermo Fisher). All cultures were maintained at 37 °C and 5% CO₂. For induction of DNA damage, cells were treated with camptothecin (CPT, 1 µM, Selleck) for 1 h, etoposide (ETO, 100 μg/mL, Selleck) for 4 h, or γ-irradiation. ATM inhibitor (ATMi, KU-55933), ATR inhibitor (ATRi, VE-821) and DNA-PK inhibitor (DNA-PKi, NU7441) were purchased from Selleck. During DNA damage induction by ionizing radiation (IR), CPT or ETO, the corresponding inhibitors were maintained in complete medium continuously unless indicated.

Chen Y., Shen H., Liu T., Cao K., Wan Z., Du Z., Wang H., Yu Y., Ma S., Lu E., Zhang W., Cai J., Gao F, & Yang Y. (2023). ATR-binding lncRNA ScaRNA2 promotes cancer resistance through facilitating efficient DNA end resection during homologous recombination repair. Journal of Experimental & Clinical Cancer Research : CR, 42, 256.

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Hematopoietic Stem Cell Culture Protocol

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Evaluating DNA Damage Checkpoint Inhibitors

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The following drugs and checkpoint inhibitors were used: Hydroxyurea (Calbiochem), Aphidicolin (Acros Organics), Camptothecin (Calbiochem), ATRi VE-821 (Selleckchem), CHK1i LY2603618 (Selleckchem), ATMi KU-55933 (Selleckchem), DNA-PKcsi NU7441 (Selleckchem).

Menolfi D., Jiang W., Lee B.J., Moiseeva T., Shao Z., Estes V., Frattini M.G., Bakkenist C.J, & Zha S. (2018). Kinase-dead ATR differs from ATR loss by limiting the dynamic exchange of ATR and RPA. Nature Communications, 9, 5351.

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