Aphidicolin (Sigma, Saint Louis, MO, USA, A0781) (5 mg/mL, 24 h) was used to synchronize BmN-SWU1 cells in the G1 phase. Hydroxyurea (Sigma, Saint Louis, MO, USA, H8627) was used to synchronize BmN-SWU1 cells in the S phase. Cells were incubated with 1 mM Hydroxyurea for 16 h and cultured in Hydroxyurea-washed medium for 7 h. Nocodazole (Sigma, Saint Louis, MO, USA, M1404) (10 mg/mL, 24 h) was used to synchronize BmN-SWU1 cells in the G2/M phase.
Hydroxyurea
Manufactured by Merck Group
Sourced in United States, Germany, France, Italy, United Kingdom
Hydroxyurea is a chemical compound used in various laboratory applications. It functions as an inhibitor of ribonucleotide reductase, an enzyme involved in the synthesis of DNA.
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Lab products found in correlation
Aphidicolin, by Merck Group (38 mentions)
Nocodazole, by Merck Group (25 mentions)
Camptothecin, by Merck Group (22 mentions)
Etoposide, by Merck Group (20 mentions)
Cycloheximide (chx), by Merck Group (19 mentions)
Cisplatin, by Merck Group (19 mentions)
Thymidine, by Merck Group (17 mentions)
Mitomycin c, by Merck Group (16 mentions)
Olaparib, by Selleck Chemicals (12 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (11 mentions)
Doxycycline, by Merck Group (11 mentions)
Methyl methanesulfonate, by Merck Group (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (9 mentions)
Bromodeoxyuridine (brdu), by Merck Group (9 mentions)
Gemcitabine, by Merck Group (8 mentions)
Mg132, by Merck Group (8 mentions)
Puromycin, by Merck Group (8 mentions)
Ve 821, by Selleck Chemicals (8 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (8 mentions)
Streptomycin, by Thermo Fisher Scientific (7 mentions)
356 protocols using hydroxyurea
1
Cell Synchronization Techniques in BmN-SWU1
2
Labeling Proliferative Cells in Fish
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To labelling proliferative cells, fish were transferred to 0.2 mM EdU (900584, Sigma-Aldrich) for 1.5 h before collecting samples. Three individuals were treated in a minimum of 120 ml of freshly prepared EdU solution (in system water).
In hydroxyurea treatment, fish were preincubated in 50 mM hydroxyurea (400046, Sigma-Aldrich) solution (in system water) for 3 h, anesthetized (0.02% MS-222), and subjected to corneal abrasion, then kept in the hydroxyurea solution for 3 h before sample collection. For TGF-beta receptor I inhibition, fish were preincubated with 50 µM SB431542 (S4317, Sigma-Aldrich) solution (in system water) for 3 h, anesthetized (0.02% MS-222), and subjected to corneal abrasion, then kept in the inhibitor solution for 3 h before sample collection.
In hydroxyurea treatment, fish were preincubated in 50 mM hydroxyurea (400046, Sigma-Aldrich) solution (in system water) for 3 h, anesthetized (0.02% MS-222), and subjected to corneal abrasion, then kept in the hydroxyurea solution for 3 h before sample collection. For TGF-beta receptor I inhibition, fish were preincubated with 50 µM SB431542 (S4317, Sigma-Aldrich) solution (in system water) for 3 h, anesthetized (0.02% MS-222), and subjected to corneal abrasion, then kept in the inhibitor solution for 3 h before sample collection.
3
Hydroxyurea-induced cell cycle arrest
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Capsaspora cells were incubated at 23°C in ATCC medium 1034 (modified PYNFH medium). Two cultures of Capsaspora independently propagated (coming from a culture split two passagings before) at 30–50% confluency were treated using 10mM Hydroxyurea (Sigma Aldrich, Saint Louis, MO, USA, #H8627) in culture medium, and left incubating for approximately 14 hours. After treatment, cells were washed by: (i) centrifuging at 5000G for 5’, (ii) discarding the supernatant, (iii) resuspending in 15ml of fresh medium, (iv) centrifuging at 5000G for 5’, (v) discarding supernatant, (vi) eluting in fresh medium for a final cell density of ~1 million cells/ml. From this, cells were seeded in 5ml flask cultures for time-point sampling. Samples were collected by scraping and washing two hours after release, and from there on every 45 minutes until thirteen hours. A total of sixteen time points were taken, constituting a time window comprising one event of genome duplication and one mitotic division.
We also tested different concentrations and incubation times of Hydroxyurea, nocodazole (Sigma-Aldrich, #M1404), and aphidicolin (Sigma-Aldrich, #A0781). Only 10mM Hydroxyurea for longer than thirteen hours showed arrest of the cell cycle, while nocodazole had no observable effect by DNA content measurement, and the rest ruined the samples due to insolubility of the compound.
We also tested different concentrations and incubation times of Hydroxyurea, nocodazole (Sigma-Aldrich, #M1404), and aphidicolin (Sigma-Aldrich, #A0781). Only 10mM Hydroxyurea for longer than thirteen hours showed arrest of the cell cycle, while nocodazole had no observable effect by DNA content measurement, and the rest ruined the samples due to insolubility of the compound.
4
Synchronized Cell Cycle Analysis
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Log-phase cultures were synchronized using hydroxyurea (Sigma-Aldrich) added to a final concentration of 0.2 M for 1.5 h. hydroxyurea was then rinsed out with a series of three washes with SC media, and the resuspended cultures were incubated at 37°C for 1 h. For nocodazole treatment, log-phase cultures in SC were incubated at 37°C for 1 h before supplementation with 10% pre-warmed YPD media. The culture was split and treated with either 25 µg/ml nocodazole and 1% DMSO, or 1% DMSO alone. Cultures were incubated another 1.5 h at 37°C and then imaged live at 37°C. Imaging conditions were maintained at 300-ms exposure for CFP, and 500-ms exposure for YFP fluorescence over 14 Z-planes spaced 0.5 µm apart. For analysis, the intensity of all images was adjusted equally using SlideBook.
5
Semi-Synthetic Anticancer Diterpenoid Derivatives
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7α-acetoxy-6β-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz; Fig. 1a ) was obtained by semi-synthesis from the natural diterpenoid 7α-acetoxy-6β-hydroxyroyleanone, isolated from a Lamiaceae family plant, as described in ref. 43 (link). PMA and ARA were purchased from Enzo Life Science (Grupo Taper SA, Sintra, Portugal). ETOP, hydroxyurea, and cyclophosphamide (CP) were obtained from Sigma-Aldrich (Sintra, Portugal). All compounds were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich), except hydroxyurea and CP that were dissolved in water.
6
Cell Cycle Profiling: Flow Cytometry
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Cell cycle profiling using PI/RNase Staining Buffer (BD Biosciences, San Jose, CA, USA) was performed according to the manufacturer’s instructions. Phase distribution of the cell cycle was examined after 18 h with 100 nM etoposide, 5 mM hydroxyurea, 50 ng/mL nocodazole, 10 nM paclitaxel, or a double 2 mM thymidine block for 18 h and released for 6 h. etoposide, hydroxyurea, nocodazole, paclitaxel, and thymidine were purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). All analytic cytometry procedures were performed on a FACS Canto II flow cytometer (Becton–Dickinson, Franklin Lakes, NJ, USA). The Watson pragmatic fitting algorithm was used to determine cell cycle phase statistics using FlowJo v10.
7
Cell Synchronization Techniques for Mitosis Analysis
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We used several methods for cell synchronization. To synchronize cells at the G1/S transition, we applied thymidine-hydroxyurea blocks. HeLa cells were pre-synchronized in S phase by incubation with 2.0 mM thymidine (Sigma-Aldrich) for 20 h, released in drug-free medium for 8 h, then cells were resynchronized at the G1/S transition by incubation with 1.5 mM hydroxyurea (Sigma-Aldrich) for 13 h. When needed, plasmid DNA was transfected at 2 h after release from thymidine. The G1/S cells were then washed, incubated in fresh medium for the appropriate time, and used for experiments.
For mitotic time course analysis, cells were arrested at prometaphase using hydroxyurea-monastrol or hydroxyurea-nocodazole blocks. The cells were pre-synchronized at the G1/S transition by incubation with 1.5 mM hydroxyurea for 20 h, released for 3 h and then synchronized at the early phase of mitosis by treatment with medium containing 100 nM monastrol (Sigma-Aldrich), an inhibitor of the mitotic kinesin Eg5 (Kapoor et al., 2000 (link)) or 100 ng/ml nocodazole for 6 h. Cells at prometaphase were collected by the shake-off method (Jackman and O'Connor, 2001 (link)), aliquoted, and cultured in fresh medium for the indicated period. The cells were then immediately lysed in sample buffer.
For mitotic time course analysis, cells were arrested at prometaphase using hydroxyurea-monastrol or hydroxyurea-nocodazole blocks. The cells were pre-synchronized at the G1/S transition by incubation with 1.5 mM hydroxyurea for 20 h, released for 3 h and then synchronized at the early phase of mitosis by treatment with medium containing 100 nM monastrol (Sigma-Aldrich), an inhibitor of the mitotic kinesin Eg5 (Kapoor et al., 2000 (link)) or 100 ng/ml nocodazole for 6 h. Cells at prometaphase were collected by the shake-off method (Jackman and O'Connor, 2001 (link)), aliquoted, and cultured in fresh medium for the indicated period. The cells were then immediately lysed in sample buffer.
8
Inducing Miniature Tissues in Zebrafish
9
Morphology and Migration of NRAS Mutant Cells
10
Cell Cycle Synchronization Protocols
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S phase arrest was achieved by synchronizing the cells in G1 with α factor and then releasing them into SC medium with 200 µM hydroxyurea (Sigma; H8726). For cells harboring the CUP1prCDC5 construct and their respective controls, hydroxyurea was added to asynchronous cultures, since adding the drug to α factor–synchronized cells was slightly toxic. 100 µM copper sulfate (Sigma; 209198) was added after to cells after 2 h in hydroxyurea. To achieve arrest in M phase, cells were synchronized in G1 and released into cell cycle without CDC20, an activator of the anaphase-promoting complex. CDC20 was under a methionine-repressible MET3 promotor. Therefore, cells were grown and synchronized in medium lacking methionine but released into medium with methionine to block Cdc20 expression.
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