Icrf 159

Manufactured by Merck Group

ICRF-159 is a laboratory equipment product manufactured by Merck Group. It is a chemical compound used in scientific research and analysis. The core function of ICRF-159 is to serve as a research tool for investigating various biological processes and pathways.

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Lab products found in correlation

Hydroxyurea, by MP Biomedicals (1 mentions) Aphidicolin, by Thermo Fisher Scientific (1 mentions) Chk1i azd7762, by Selleck Chemicals (1 mentions) Atmi ku 55933, by Selleck Chemicals (1 mentions) Lipofectamine ltx, by Thermo Fisher Scientific (1 mentions) Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions) Nocodazole, by Fujifilm (1 mentions) Round cover glasses, by Matsunami (1 mentions) Neocarzinostatin ncs, by Merck Group (1 mentions) Paraformaldehyde (pfa), by Merck Group (1 mentions) Etoposide, by Merck Group (1 mentions) Thymidine, by Merck Group (1 mentions) Aphidicolin, by Merck Group (1 mentions) Amniomax c 100 complete medium, by Thermo Fisher Scientific (1 mentions) Rnaimax, by Thermo Fisher Scientific (1 mentions) Neon transfection system, by Thermo Fisher Scientific (1 mentions)

4 protocols using icrf 159

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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Detecting Anaphase Bridges in HeLa Cells

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HeLaS3 cells grown on 13 mm–diameter round cover glasses (Matsunami) were arrested at M phase by treatment with 0.1 µg/ml nocodazole (Wako) for 3 h. Etoposide (Sigma-Aldrich) or neocarzinostatin (NCS, Sigma-Aldrich) or ICRF-159 (Sigma-Aldrich) was added to the culture medium to a final concentration of 10 µM, 1 ng/ml, 10 µM, respectively. Cells were incubated for 15 min in medium containing the drug and then washed twice with PBS. For detection of anaphase bridges, Etoposide-treated cells were incubated in fresh culture medium for 1 h, fixed with 4% (w/v) paraformaldehyde (Sigma-Aldrich), and stained with 4′,6-Diamidino-2-Phenylindole (DAPI). The frequency of cells with at least one bridge was calculated by dividing the number of cells containing bridges by the number of total anaphase cells.

Terasawa M., Shinohara A, & Shinohara M. (2014). Canonical Non-Homologous End Joining in Mitosis Induces Genome Instability and Is Suppressed by M-phase-Specific Phosphorylation of XRCC4. PLoS Genetics, 10(8), e1004563.

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ICRF-159 and Thymidine Treatment of HeLa Cells

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HeLa cells were cultured in DMEM supplemented with 10% FCS as previously described [16 (link)].
ICRF-159 (Razoxane) was provided by Sigma Aldrich (R8657) and was added to the cell culture medium at a final concentration of 1 or 10 µM following the protocol described in figures 1a, 2c, 3a, 4a and electronic supplementary material, figure S2A. Thymidine was provided by Sigma Aldrich (T9250) and was added to the cell culture medium at a final concentration of 2 mM.
All cells were routinely checked for mycoplasma infection.

Gemble S., Buhagiar-Labarchède G., Onclercq-Delic R., Fontaine G., Lambert S, & Amor-Guéret M. (2020). Topoisomerase IIα prevents ultrafine anaphase bridges by two mechanisms. Open Biology, 10(5), 190259.

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Culturing and Transfecting Fibroblast Cell Lines

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Dermal fibroblasts were obtained by skin punch biopsy and cultured in amnioMAX C-100 complete medium (Life Technologies). MEFs were prepared from individual E13.5 embryos after removing the head and abdominal cavities. Each embryo was minced and maintained in DMEM, 10% FCS, 0.1 mM β-mercaptoethanol, 100 U/mL penicillin, and 100 µg/mL streptomycin. All fibroblast cultures were maintained in a 37°C incubator with 5% CO₂ and 3% O₂. hTERT RPE-1 cells were obtained from the American Type Culture Collection (CRL-4000) and cultured in DMEM:F12, 10% FCS, 0.26% sodium bicarbonate, 100 U/mL penicillin, and 100 µg/mL streptomycin. All cell lines were routinely tested for mycoplasma. siRNA oligonucleotides were transfected into hTERT RPE1 cells using RNAiMAX (Life Technologies) according to the manufacturer's instructions. For siRNA oligonucleotide sequences, see Supplemental Table S2C. pRFP-H2B was transfected into primary fibroblast cells by electroporation with the Neon transfection system (Thermo Fischer Scientific) according to the manufacturer's instructions. Where indicated, cells were treated with 0.3 µM aphidicolin (Sigma-Aldrich) or 5 µM ICRF-159 (Sigma-Aldrich) for 24 h.

Martin C.A., Murray J.E., Carroll P., Leitch A., Mackenzie K.J., Halachev M., Fetit A.E., Keith C., Bicknell L.S., Fluteau A., Gautier P., Hall E.A., Joss S., Soares G., Silva J., Bober M.B., Duker A., Wise C.A., Quigley A.J., Phadke S.R., Wood A.J., Vagnarelli P, & Jackson A.P. (2016). Mutations in genes encoding condensin complex proteins cause microcephaly through decatenation failure at mitosis. Genes & Development, 30(19), 2158-2172.

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