Nu6027

Manufactured by Merck Group

NU6027 is a laboratory equipment product. It serves as a core function, but a detailed description while maintaining an unbiased and factual approach is not available.

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Lab products found in correlation

Anti myc, by Fortis Life Sciences (2 mentions) Etoposide, by Merck Group (2 mentions) Dasatinib, by Selleck Chemicals (1 mentions) Mifepristone, by Merck Group (1 mentions) Mk 1775, by Selleck Chemicals (1 mentions) Chir 124, by Selleck Chemicals (1 mentions) Phospho ser thr atm atr substrate antibody, by Cell Signaling Technology (1 mentions) Anti gst, by GenScript (1 mentions) Hydroxyurea, by Merck Group (1 mentions) Anti usp20, by Fortis Life Sciences (1 mentions) Anti claspin, by Fortis Life Sciences (1 mentions) Anti atr, by Fortis Life Sciences (1 mentions) Anti flag m2 f1804, by Merck Group (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Bromodeoxyuridine (brdu), by BD (1 mentions) Monobromobimane, by Merck Group (1 mentions) Diallyl trisulfide, by Cayman Chemical (1 mentions) Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions) Anti β actin, by Abcam (1 mentions) Rabbit anti gadph, by Merck Group (1 mentions)

7 protocols using nu6027

Pharmacologically-Active Compound Library

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The Library of Pharmacologically-Active Compounds (LOPAC), 6α-methylprednisolone (6MP), dexamethasone (Dex), mifepristone, PD166285 (PD16), PD173952 (PD17), NU6027 and CGP7454A were purchased from Sigma-Aldrich; dasatinib, MK-1775 and CHIR-124 from SelleckChem. All compounds were diluted in DMSO to 10 mM stock solutions and kept at -20°C.

Akbib S., Stichelmans J., Stangé G., Ling Z., Assefa Z, & Hellemans K.H. (2019). Glucocorticoids and checkpoint tyrosine kinase inhibitors stimulate rat pancreatic beta cell proliferation differentially. PLoS ONE, 14(2), e0212210.

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Characterization of DNA Damage Response Pathways

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Hydroxyurea (HU, a final concentration of 2 mM was used throughout this study), cycloheximide (CHX, a final concentration of 50 μg/ml was used) and the ATR inhibitor NU6027 (a final concentration of 10 μM was used), were purchased from Sigma. BrdU (a final concentration of 20 μM was used) was from BD Biosciences.
Rabbit polyclonal antibodies used for immunoblotting and/or immunoprecipitaion including anti-MYC (A190–205A), anti-HA (A190–208A), anti-USP20 (A301–189A), anti-CLASPIN (A300–267A), anti-HERC2 (A301–905A), anti-ATR (A300–138A) were from the Bethyl Laboratories; Chk1 antibody (sc-8408) was from the Santa Cruz Biotechnology. Rabbit monoclonal antibody anti-GST (A00865) was from the GenScript. Mouse monoclonal antibody anti-FLAG M2 (F1804) was from Sigma. Phospho-Chk1 (Ser345) (Rabbit mAb #2348) and Phospho-(Ser/Thr) ATM/ATR substrate antibody (#2851) were from Cell Signaling. Anti-BrdU fluorescein isothiocyanate (FITC) (347583) used for immunofluorescence was from BD Biosciences.
The detailed information of all the expression constructs is available upon request.
All cell lines were cultured in high-glucose Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37°C.

Zhu M., Zhao H., Liao J, & Xu X. (2014). HERC2/USP20 coordinates CHK1 activation by modulating CLASPIN stability. Nucleic Acids Research, 42(21), 13074-13081.

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Quantifying Redox-Sensitive Protein Modifications

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Monobromobimane (MBB), Tris (2-carboxyethyl)phosphine hydrochloride (TCEP), sulfosalicylic acid (SSA), 1-fluoro-2,4-dinitrobenzene (DNFB), TPP® tissue culture dishes, NU6027, penicillin/streptomycin, and t-BOOH were purchased from Sigma (St. Louis, MO). Fetal bovine serum, Dulbecco’s modified Eagle’s medium (DMEM) (standard liquid media) were from Invitrogen (Rockville, MD). β-cyano-L-alanine and diallyl trisulfide were purchased from the Cayman Chemical Company (Ann Arbor, MI). Antibodies used were anti-ATR-pS435 (Cell Signaling Technology Inc, Danvers, MA, catalog number 2853s), anti-total-ATR (Invitrogen, Waltham, MA USA, catalog number PA1–450), anti-CHK1-pS345 (Invitrogen, catalog number PA5–34625), anti-total-CHK1(Abcam, Cambridge, MA, catalog number ab47574), anti-3-MST (Santa Cruz Biotechnology, Santa Cruz, CA, catalog number sc-135993), anti-CSE (Santa Cruz Biotechnology, catalog number sc-101924), anti-CBS (Santa Cruz Biotechnology, catalog number sc-67154), and anti-Visfatin (Nampt, Bethyl Laboratories, Montgomery TX, catalog number A300–779A), rabbit anti-GADPH (Sigma, catalog number G9545), and anti-beta-actin (abcam, catalog number ab8227). Secondary antibodies were goat anti-rabbit IgG (catalog numbers ab6721 & ab2040, Abcam), goat anti-mouse IgG (catalog number ab205719, Abcam), and goat anti-mouse IgG (catalog number sc358920, Santa Cruz).

Chen J., Shen X., Pardue S., Meram A.T., Rajendran S., Ghali G.E., Kevil C.G, & Shackelford R.E. (2018). The Ataxia telangiectasia-mutated and Rad3-related Protein Kinase Regulates Cellular Hydrogen Sulfide Concentrations. DNA repair, 73, 55-63.

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Cytoprotective Screening Against Oxidative Stress

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Cells were exposed to glutamate, H₂O₂ (Sigma), sodium nitroprusside (SNP, a donor of nitric oxide, Sigma), and ZnCl₂ (Sigma) in minimum essential media (Gibco) for 24 h at the indicated concentrations to induce cell death. Anthranilic acid, clotrimazole, dantrolene, flufenamic acid, kenpaullone, NU6027, olomoucine, roscovitine, ruthenium red, SU9516, N,N,N,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), and 2-aminoethyl diphenylborinate (2-APB) were purchased from Sigma. Capsaicin, MK-801, ML204, Pyr3, and (−)-Xestospongin C (XeC) were purchased from Tocris. SB216763 was purchased from Enzo Life Science, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was purchased from RBI. All reagents were added 1 h prior to H₂O₂ exposure, unless otherwise stated.

Park S.E., Song J.H., Hong C., Kim D.E., Sul J.W., Kim T.Y., Seo B.R., So I., Kim S.Y., Bae D.J., Park M.H., Lim H.M., Baek I.J., Riccio A., Lee J.Y., Shim W.H., Park B., Koh J.Y, & Hwang J.J. (2018). Contribution of Zinc-Dependent Delayed Calcium Influx via TRPC5 in Oxidative Neuronal Death and its Prevention by Novel TRPC Antagonist. Molecular Neurobiology, 56(4), 2822-2835.

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Inducing DNA Damage and Cell Cycle Arrest

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To generate DNA damage, HeLa cells were treated with 0.5 µM B[a]P (Sigma) and 2.5 µM B[a]P-7,8-diol-9,10-epoxide (Sigma) for 48 h or 0.5 µM etoposide (Sigma) for 24 h. For the inhibition experiments, cells were incubated with the ATR inhibitor NU6027 (2 µM) (Sigma), the ATM inhibitor KU55933 (20 nM) (Sigma), (Enzo Life Sciences), the Chk1 inhibitor UCN-01 (300 nM) (Sigma), the Chk2 inhibitor (1 µM), and the DNA-PK inhibitor (0.5 µM) for 24 h. For synchronization in G2, cells were treated with the Cdk1 inhibitor RO3306 (1 µM) for 24 h. For inhibition of mitotic kinases, cells were treated with the Cdk1 inhibitor RO3306 (1 µM) for 5 h, the Plk1 inhibitor BI2536 (1 µM) for 1 h, the Aurora A inhibitor VX680 (5 µM) for 1 h, and the Aurora B inhibitor Hesperadin (2 µM) (Selleckem) for 5 h. Mitotic arrest was achieved by the treatment of 100 ng/ml nocodazole for 16 h.

Song H., Kim E.H., Hong J., Gwon D., Kim J.W., Bae G.U, & Jang C.Y. (2023). Hornerin mediates phosphorylation of the polo-box domain in Plk1 by Chk1 to induce death in mitosis. Cell Death and Differentiation, 30(9), 2151-2166.

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Detailed Protocols for DNA Damage Response

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The ATM inhibitor KU55933 (a final concentration of 10 μM was used throughout this research) was purchased from Selleck, and the DNA-PKcs inhibitor NU7026 (a final concentration of 10 μM was used), the ATR inhibitor NU6027 (a final concentration of 10 μM was used), and etoposide (a final concentration of 10 μM was used) were purchased from Sigma.
Rabbit polyclonal antibodies anti-BCL10, anti-MALT1, anti-DNA-PKs, anti-BRCA1, anti-MYC, anti-HA, and anti-RAP80 were from Bethyl; mouse monoclonal antibodies anti-γ-H2AX, rabbit polyclonal antibodies anti-RNF168 (ABE367) for IF, anti-RNF168 (06-1130) for IP, and anti-ubiquitin (FK2) were from Millipore; rabbit monoclonal antibody anti-GST (A00865) was from GenScript; mouse monoclonal antibody anti-FLAG (M2) and rabbit polyclonal antibody anti-BCL10 (MK-17 for IF) were from Sigma; mouse monoclonal antibody anti-BRCA1 (D-9) and anti-RNF168 (B-11) for IB were from Santa Cruz. Rabbit polyclonal anti-MDC1 antibody was described before.¹¹ (link) Rabbit polyclonal antibodies against RNF8, RAD51, and UBC13 were gifts from Drs Michael SY Huen, Jun Huang, and Wei Xiao, respectively. The rabbit polyclonal anti-pT91-BCL10 antibody was raised against the phospho-peptide IRREK(pT)QNFLI and affinity-purified (AbMart).
All cell lines were cultured in high-glucose Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum at 37 °C.

Zhao H., Zhu M., Dou G., Zhao H., Zhu B., Li J., Liao J, & Xu X. (2014). BCL10 regulates RNF8/RNF168-mediated ubiquitination in the DNA damage response. Cell Cycle, 13(11), 1777-1787.

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Dexamethasone and Luciferase Assay

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Dexamethasone (Dex), PMA (phorbol myristate acetate), NU6027, and phenanthroline were purchased from Sigma (St. Louis, MO). Restriction enzymes and T4 DNA ligase were from New England Biolabs (Beverly, MA) and the dual-luciferase reporter assay was from Promega (Madison, WI).

Chow C.C., Finn K.K., Storchan G.B., Lu X., Sheng X, & Simons SS J.r. (2015). Kinetically-Defined Component Actions in Gene Repression. PLoS Computational Biology, 11(3), e1004122.

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