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Aphidicolin

Manufactured by Santa Cruz Biotechnology
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Aphidicolin is a naturally occurring tetracyclic diterpene compound. It functions as a DNA synthesis inhibitor by selectively blocking the activity of DNA polymerase alpha, which is essential for DNA replication.

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Lab products found in correlation

Aicar, by Merck Group (2 mentions) Neocarzinostatin, by Merck Group (2 mentions) Azd1775, by Selleck Chemicals (2 mentions) Az3146, by Santa Cruz Biotechnology (2 mentions) Ro3306, by Cayman Chemical (2 mentions) Actinomycin d, by Merck Group (2 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Chloroquine, by Merck Group (1 mentions) Camptothecin, by Merck Group (1 mentions) Bmh 21, by Merck Group (1 mentions) Marizomib, by Merck Group (1 mentions) Cx 5461, by Selleck Chemicals (1 mentions) Quarfloxin, by Adooq (1 mentions) Talazoparib, by Axon Medchem (1 mentions) Penicillin streptomycin, by Merck Group (1 mentions) Pf 573228, by Cayman Chemical (1 mentions) Advanced dulbecco s modified eagle medium f12, by Thermo Fisher Scientific (1 mentions) Aphidicolin, by Cayman Chemical (1 mentions) Dulbecco s modified eagle medium f12, by Merck Group (1 mentions) Mcilwain tissue chopper, by Ted Pella (1 mentions)

8 protocols using aphidicolin

1

Cell Cycle Regulation and DNA Damage

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Where indicated, cells were treated with aphidicolin (Santa Cruz); RO3306 (Cayman Chemical); B02 (Cayman Chemical); AICAR (Sigma-Aldrich); AZD1775 (Selleckchem); AZ3146 (Santa Cruz); Neocarzinostatin (Sigma-Aldrich) at the indicated concentrations and for the indicated lengths of time.
Wassing I.E., Graham E., Saayman X., Rampazzo L., Ralf C., Bassett A, & Esashi F. (2021). The RAD51 recombinase protects mitotic chromatin in human cells. Nature Communications, 12, 5380.
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2

Cytotoxic Drugs Screening Assay

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The following drugs were used: camptothecin (CPT), actinomycin D, cycloheximide, marizomib, chloroquine, Bmh21, BO-2, (all Sigma-Aldrich), aphidicolin (Santa Cruz), talazoparib (Axon Medchem), quarfloxin (CX-3543, Adooq Bioscience) and CX-5461 (Selleckchem).
van Schie J.J., Faramarz A., Balk J.A., Stewart G.S., Cantelli E., Oostra A.B., Rooimans M.A., Parish J.L., de Almeida Estéves C., Dumic K., Barisic I., Diderich K.E., van Slegtenhorst M.A., Mahtab M., Pisani F.M., te Riele H., Ameziane N., Wolthuis R.M, & de Lange J. (2020). Warsaw Breakage Syndrome associated DDX11 helicase resolves G-quadruplex structures to support sister chromatid cohesion. Nature Communications, 11, 4287.
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3

Molar Development ex vivo Explant Culture

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Frontal slices containing tooth germs of E13.5, E14.0, and E14.5 mandibular molars were obtained as described (Alfaqeeh and Tucker 2013 ). Briefly, mandibles were manually dissected from the heads in Advanced Dulbecco’s Modified Eagle Medium F12 (Gibco) and sliced frontally into 200-μm sections with a McIlwain Tissue Chopper (Ted Pella, Inc.). Slices were cultured on PET membranes (353090; Corning) on a steel mesh (FE228710; Goodfellow) in Advanced Dulbecco’s Modified Eagle Medium F12 with 1% penicillin-streptomycin (P4333; Sigma), 15% fetal calf serum, and 0.1 g/L of vitamin C at 37 °C in 5% CO2 humidified atmosphere. Explants were treated with aphidicolin (2.0 μg/mL in dimethyl sulfoxide [DMSO]; Santa Cruz Biotechnology); 10μM BrdU was added after 3 or 23 h; and explants were fixed at 5 or 25 h in 4% paraformaldehyde (PFA) for 4 h at room temperature. Explants were photographed under a stereo zoom dissection microscope with brightfield optics at the 3- and 23-h time points (to avoid the trivial shape perturbation sometimes caused by addition of the BrdU-containing medium). For FAK inhibition, explants were treated with 1µM PF-573228 (Cayman Chemicals) in place of aphidicolin.
Yamada S., Lav R., Li J., Tucker A.S, & Green J.B. (2019). Molar Bud-to-Cap Transition Is Proliferation Independent. Journal of Dental Research, 98(11), 1253-1261.
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4

Cell Line Maintenance and Reagents

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U2OS, SJSA, MCF7, SK-BR3, MDA-MB-231, ZR75-1, and A549 cells were obtained from the American Type Culture Collection (ATCC) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum (FBS). HCT116 p53+/+ and p53−/− cells were kindly provided by Professor Bert Vogelstein and maintained in McCoy’s 5 A medium containing 10% FBS. LNCap, DU145, and PC3 cells were obtained from ATCC and maintained in RPMI-1640 medium containing 10% FBS. The following chemicals were obtained from commercial sources: bleomycin (HY-17565; MCE, Monmouth Junction, NJ, USA), mitomycin C (HY-13316; MCE), hydroxyurea (HY-B0313; MCE), methotrexate (HY-14519; MCE), cyclophosphamide (HY-17420; MCE), teniposide (HY-13761; MCE), nutlin-3 (HY-50696; MCE), aphidicolin (sc-201535A; Santa Cruz Biotechnology, Dallas, TX, USA), doxorubicin (S1208; Selleck, Houston, TX, USA), camptothecin (S1288; Selleck), paclitaxel (S1150; Selleck), cisplatin (P4394; Sigma-Aldrich, St Louis, MO, USA), and actinomycin D (129935; Sigma-Aldrich).
Cui D., Dai X., Gong L., Chen X., Wang L., Xiong X, & Zhao Y. (2020). DEPTOR is a direct p53 target that suppresses cell growth and chemosensitivity. Cell Death & Disease, 11(11), 976.
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5

Cell Transfection with Aphidicolin

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Cells were seeded and used for transfections the next day. Cells were transfected with 500 ng DNA per 100 000 cells using Lipofectamine 2000 (Thermo Fisher Scientific, UK) according to the manufacturer’s guidelines. Four hours post-transfection, 5 µg ml−1 Aphidicolin (Santa Cruz Biotechnology, USA) was added.
Loew L., Goonawardane N., Ratcliff J., Nguyen D, & Simmonds P. (2020). Use of a small DNA virus model to investigate mechanisms of CpG dinucleotide-induced attenuation of virus replication. The Journal of General Virology, 101(11), 1202-1218.
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6

DNA Damage Response Assay Reagents

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Potassium bromate KBrO3 (Sigma catalog no. 309087; CAS: 7758-01-2), sodium azide NaN3 (Fisher Chemical catalog no. S227I; CAS: 26628-22-8), ATMi KU60019 (Selleckchem catalog no. S1570), Cdk1 inhibitor IV, RO-3306 (Millipore catalog no. 217699), ETP (Cell Signaling catalog no. 2200; CAS 33419-42-0), Aphidicolin (Santa Cruz catalog no. sc-201535; CAS 38966-21-1).
Barnes R.P., de Rosa M., Thosar S.A., Detwiler A.C., Roginskaya V., Van Houten B., Bruchez M.P., Stewart-Ornstein J, & Opresko P.L. (2022). Telomeric 8-oxo-guanine drives rapid premature senescence in the absence of telomere shortening. Nature Structural & Molecular Biology, 29(7), 639-652.
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7

Inducing Replication Stress in Cells

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For induction of replication stress, cells were seeded at ~200K per 6-well plated and treated for 24 hours with 0.4 μM aphidicolin (APH) (sc-201535, Santa Cruz) dissolved in DMSO. Cells were then harvested at the indicated time points and processed for flow cytometry or exo-FISH. When performing the serum starvation time course, technical variations of exo-FISH sample preparation was minimized by harvest samples at the same point. To this end, hTERT-RPE1 cells were seeded in serial dilutions and the media was changed to serum-starved media (0.1% FBS (F9665, Merck), 1% penicillin-streptomycin (15140122, Life Technologies) and 0.123% sodium bicarbonate (S8761, Sigma-Aldrich)) at the indicated time points. Cells were split if they approached confluence before serum starvation.
Saayman X., Graham E., Nathan W., Nussenzweig A, & Esashi F. (2023). Centromeres as universal hotspots of DNA breakage, driving RAD51-mediated recombination during quiescence. Molecular cell, 83(4), 523-538.e7.
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8

Cell Cycle Regulation Assays

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Where indicated, cells were treated with aphidicolin (Santa Cruz); RO3306 (Cayman Chemical); B02 (Cayman Chemical); AICAR (Sigma-Aldrich); AZD1775 (Selleckchem); AZ3146 (Santa Cruz); Neocarzinostatin (Sigma-Aldrich) at the indicated concentrations and for the indicated lengths of time.
Wassing I.E., Saayman X., Rampazzo L., Ralf C., Bassett A., & Esashi F. (2020). The RAD51 recombinase protects mitotic chromatin in human cells.
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