5-Iodo-2′-deoxyuridine (IdU; Sigma-Aldrich, St. Louis, MO) was resuspended in sterile drinking water at a concentration of 1 g/L. Fresh IdU drinking water was provided weekly, for 4 weeks, in light protected water bottles.
5 iodo 2 deoxyuridine
Manufactured by Merck Group
Sourced in United States
5-Iodo-2′-deoxyuridine is a synthetic nucleoside analogue that can be used as a chemical compound for research purposes. It contains a halogenated pyrimidine base, 5-iodouracil, attached to a deoxyribose sugar.
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Lab products found in correlation
5 chloro 2 deoxyuridine, by Merck Group (12 mentions)
Mitomycin c, by Merck Group (3 mentions)
5 chloro 2 deoxyuridine cldu, by Merck Group (3 mentions)
Aphidicolin, by Merck Group (2 mentions)
Olaparib, by Selleck Chemicals (2 mentions)
Hydroxyurea, by Merck Group (2 mentions)
Camptothecin, by Merck Group (2 mentions)
Cisplatin, by Merck Group (2 mentions)
H8627, by Merck Group (1 mentions)
Fv1000 confocal microscope, by Olympus (1 mentions)
Nb500 169, by BD (1 mentions)
Fv1000, by Olympus (1 mentions)
Hydroxyurea, by Selleck Chemicals (1 mentions)
Monastrol, by Selleck Chemicals (1 mentions)
Genistein, by Merck Group (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Auxin, by Merck Group (1 mentions)
Abemaciclib, by Selleck Chemicals (1 mentions)
Latrunculin b, by Merck Group (1 mentions)
Mpi 0479605, by Selleck Chemicals (1 mentions)
20 protocols using 5 iodo 2 deoxyuridine
1
Chronic Administration of IdU
2
Cell Proliferation and Survival Assessment
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5-bromo-2′-deoxyuridine (BrdU; cat. no. B5002, Sigma-Aldrich, St. Louis, MO, United States) and 5-iodo-2′-deoxyuridine (IdU; cat. no. I7125, Sigma-Aldrich) were dissolved at a concentration of 15 mg/mL in sterile 0.9% NaCl solution. To assess cell survival, all rats were treated with BrdU at a dose of 50 mg/kg (i.p.) for five consecutive days during the third week of experimentation (Rivera et al., 2015 (link)). To assess cell proliferation, IdU were administered at a dose of 57.65 mg/kg (i.p.) for five consecutive days after the fifth week of experimentation (Figure 1 ).
3
DNA Fiber Assay for Replication
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The DNA fiber assay was performed as described (15 (link), 56 (link)). Cells were labeled with 50 μM 5-iodo-2′-deoxyuridine (IdU; Sigma-Aldrich, I7125) for 30 min before washing. Cells were then treated with or without HU (Sigma-Aldrich, H8627) for 4 hours. Following wash, cells were labeled with 50 μM 5-chloro-2′-deoxyuridine (CIdU; Sigma-Aldrich, C6891) for 30, 60, and 90 min, respectively. Cells were collected and suspended at a concentration of 103/μl, and 2.5 μl of cell suspension was used to prepare for the DNA fibers. Immunofluorescent staining was performed using IdU and CIdU antibodies. DNA fibers were analyzed using Olympus FV1000 confocal microscope. The lengths (1 μm = 2.59 kb) of DNA fibers were measured with the ImageJ software. At least 200 DNA fibers were examined for each sample, and each experiment was independently repeated three times.
4
Replication Dynamics in Cell Lines
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Replication fork speed and asymmetry was evaluated using the molecular combing assay. Exponentially growing cells were pretreated with DMSO, PARPi (AZD2281; 1 μM), ATRi (AZD6738; 1 μM), or the combination for 30 min. Cells were subsequently pulse-labeled with 100 μM 5-chloro-2′-deoxyuridine (CldU; cat. # C6891, Sigma-Aldrich, St. Louis, MO) followed by 100 μM 5-iodo-2′-deoxyuridine (IdU; cat. # I7125, Sigma-Aldrich, St. Louis, MO) for 15 min each, in the continuous presence of inhibitors. Afterward, cells were harvested and embedded into agarose plugs using the Genomic Vision FiberPrep® kit (Genomic Vision, Bagneux, France). DNA extraction, combing and immunostaining was performed according to the EasyComb service procedures (Genomic Vision, Bagneux, France). Coverslips were scanned with FiberVision® scanner and images were analyzed through Genomic Vision FiberStudio® software (Genomic Vision, Bagneux, France). Intact CldU (red) and IdU (green) replication tracks flanked by counterstaining were selected and reviewed for further validation.
5
Quantifying DNA Replication Dynamics
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DNA fiber analysis was performed as described (14 (link), 26 (link)). 5-Iodo-2′-deoxyuridine (IdU; 50 μM; Sigma-Aldrich, I7125) was used to label the cells for 30 min, with or without HU (Selleck, S1896) treatment, and then 250 μM 5-chloro-2′-deoxyuridine (CldU; Sigma-Aldrich, C6891) was used for the second labeling. After labeling, ~3000 cells in 2.5 μl of suspension were dropped onto one end of the glass slide, mixed with 7.5 μl of lysis buffer [50 mM EDTA, 0.5% SDS, and 200 mM tris-HCl (pH 7.5)], and incubated for 8 min at room temperature. Then, the slides were tilted to 15° to spread the DNA fibers along the slide. The slides were then treated with 2.5 M hydrochloric acid, incubated with rat anti-BrdU/CIdU (BU1/75) monoclonal antibody (Novus, NB500-169), and mouse anti-IdU monoclonal antibody (BD, 347580). The secondary antibodies were Alexa Fluor Cy3–conjugated goat anti-rat and Alexa Fluor 488–conjugated goat anti-mouse, respectively. Images were captured using a confocal microscope (Olympus, FV1000). ImageJ software was used to measure the length of DNA fibers, and the formula that 1 μm = 2.59 kb was used to convert the micrometer value to kilobase. New replication origin firing analysis was performed as described (54 ). Cells were labeled with CIdU for 10 min (without HU) or with 100 μM HU for 20 min.
6
Cellular Assays with Diverse Chemical Modulators
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The drugs were used at the following concentrations: Auxin (Sigma I5148), 500 µM; doxycycline (Sigma D3447), 2 µg ml−1; asunaprevir (Selleckchem S4935), 3 µM; monastrol (Selleckchem S8439), 50 µM; MPI-0479605 (Selleckchem S7488), 1 µM; genistein (Sigma G6649), 10 µM; SP600125 (Selleckchem S1460), 10 µM; abemaciclib (Selleckchem S5716), 50 nM or 0.5 µM; K03861 (Selleckchem S8100), 400 nM or 1 µM; palbociclib (Selleckchem S1579), 120 nM or 1 µM; aphidicolin (Sigma A0781), 0,4 µM or 1 µM; hydroxyurea (Selleckchem S1896), 2 mM; PHA767491 (Sigma PZ0178), 1 µM; RO3306 (Calbiochem 217699), 10 µM; dihydrocytochalasin D (Sigma D1641), 0,75 µM; latrunculin B (Sigma L5288), 5 µM; 5′-chloro-2′-deoxyuridine (CIdU) (Sigma C6891), 100 µM; 5′-iodo-2′-deoxyuridine (IdU) (Sigma I7125), 100 µM.
7
Cell Proliferation and Survival Analysis
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To analyze cell proliferation and survival, 5′-bromo-2′-deoxyuridine (BrdU, cat. no. B5002, Sigma-Aldrich, Sant Louis, USA) and 5′-iodo-2′-deoxyuridine (IdU, cat. no. I7125, Sigma-Aldrich) were administered intraperitoneally (i.p.) in a sterile 0.9% NaCl solution at a dose of 50 mg/kg/day and 42.75 mg/kg/day, respectively.
BrdU was administered to all experimental groups. BrdU was injected i.p. twice per day at 10 h intervals for 3 consecutive days before sacrifice. Thus, BrdU was used as a cell proliferation marker. IdU was injected i.p. twice per day at 10 h intervals for 3 consecutive days on days 2–4 of the experiment. Using this regimen of administration, IdU was administered to the control, APAPx4–6 days, and APAPx4–15 days groups only. Thus, IdU was used as a cell survival marker (Figure 9 ).
BrdU was administered to all experimental groups. BrdU was injected i.p. twice per day at 10 h intervals for 3 consecutive days before sacrifice. Thus, BrdU was used as a cell proliferation marker. IdU was injected i.p. twice per day at 10 h intervals for 3 consecutive days on days 2–4 of the experiment. Using this regimen of administration, IdU was administered to the control, APAPx4–6 days, and APAPx4–15 days groups only. Thus, IdU was used as a cell survival marker (
8
Pharmacological Inhibitors for Cancer Research
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The following chemicals or inhibitors were purchased from Selleck Chemicals (Houston, TX, USA); A-485 (Cat# S8740), Enzalutamide (Cat# S1250) and Olaparib (Cat# S1060). Etoposide (Cat# E1383), Mitomycin C (Cat# M4287), 5-Chloro-2′-deoxyuridine (Cat# C6891, 5-Iodo-2′-deoxyuridine (Cat# I7125), Dimethyl Sulfoxide (Cat# D8418) and 4′,6-Diamidino-2-phenylindole dihydrochloride (Cat# D9542) were purchased from Sigma-Aldrich (St. Louis, MO, USA). BRD9 inhibitor (Cat# T6859), SWI/SNF inhibitor, AU-15330 (T39954) and bromodomain inhibitor GNE-049 were purchased from TargetMol (Wellesley Hills, MA, USA).
9
Dual DNA Replication Labeling Assay
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Cells were sequentially labelled with 25 µM of 5-Chloro-2′-deoxyuridine (CldU) (#C6891, Sigma-Aldrich) and then 250 µM of 5-Iodo-2’-deoxyuridine (IdU) (#I7125, Sigma-Aldrich) for 30 min each [32 (link)]. Cells were resuspended in ice-cold PBS at 5 × 105 cells/mL. Four drops of 2.5 µL of cell suspension was pipetted in staggered rows onto a microscope slide (Cat #4951PLUS602811, Fisherbrand™). In all, 6 µL of spreading buffer (200 mM Tris-HCl, 50 mM EDTA, 0.5% SDS, pH 7.4) was added to cell suspension drops. DNA was allowed to run down the slide, slowly tilting slides at 15–45°. DNA was air-dried and fixed in methanol/acetic acid (3:1) for 10 min. DNA was denatured in 2.5 M HCl for 45 min. Slides were incubated in blocking solution (PBS, 2% BSA, 0.1% Tween) for 1 h, followed by rat anti-BrdU [clone BU1/75 (ICR1)] (1:100, #ab6326, Abcam) and Mouse anti-BrdU [clone B44] antibodies (1:20, #347580, BD Biosciences), overnight at 4 °C. Slides were incubated with anti-Rat AlexaFluor 568 (1:500, #A11077, Invitrogen) and anti-Mouse AlexaFluor 488 (1:500, #A21202, Invitrogen) antibodies, for 1 h at room temperature. Slides were mounted in ProLong™ Gold antifade Mountant (#P36930, Invitrogen) and analysed using a Zeiss Axio Observer Z1 microscope with ×63 oil objective. Replication fork speed was calculated using the CldU + IdU track length/60 min * 2.59 kb/µm.
10
Quantifying DNA Replication Dynamics
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To analyze origin density, cells were pulsed labeled with 25 μM 5-iodo-2′-deoxyuridine (Sigma-Aldrich) and with 25 μM 5-chloro-2′-deoxyuridine (Sigma-Aldrich). The DNA was stretched on glass slides and the incorporated nucleotides were detected by IdU antibody (mouse anti-bromodeoxyuridine, Becton Dickinson Immunocytometry Systems) and CldU antibody (monoclonal rat anti-bromodeoxyuridine [anti-BrdU], Accurate Chemical and Scientific Corporation) as described above. We used Student’s t test with two-tailed distribution for P value calculation.
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