5 chloro 2 deoxyuridine

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.

For each LCL sample, ∼100 000 cells were labelled with thymidine analogues, 20 µM 5-chloro-2′-deoxyuridine (CldU, Sigma-Aldrich, C6891) for 30 min followed by 200 µM 5-Iodo-2′-deoxyuridine (IdU, Sigma-Aldrich, I7125) for 30 min and subsequently washed thrice with PBS. Labelled cells were mixed with lysis buffer in 1:1 ratio before mounted on SuperFrost Plus slides (Epredia, J1800AMNZ). Cellular DNA were spread by slanting the slides and fixed in 3:1 methanol/acetic acid for 30 min, followed by denaturation with 2.5 N hydrochloric acid for 40 min and subsequently blocked with 2% BSA in PBS-0.1% Tween20 for 40 min at RT. Slides were washed five times with PBS followed by 2 h incubation with 1:200 anti-BrdU/IdU (Becton Dickinson, 347580, mouse) and 1:200 anti-BrdU/CldU (Abcam, ab6326, rat) antibodies. Subsequently, slides were washed five times with PBS and finally stained with anti-mouse AlexaFluor-488 (Abcam, 150157) and anti-rat AlexaFluor-594 (Abcam, 150116) conjugated secondary antibodies for 1 h at RT.
Labelled DNA fibres were imaged with 63× objective (0.103 um/pixel) using a Zeiss Inverted Fluorescence Live Cell Microscope AO7. The experiments were performed in the presence of replication stress inducing agents, such as HydroxyUrea (HU, Sigma-Aldrich, H8627) and PARP inhibitor (PARPi) (Talazoparib BMN-673, MedChem Express), as controls to assess the differences in RF speed.

Hydroxyurea (Sigma-Aldrich) was used at 2 mM; MIRIN, the inhibitor of MRE11 exonuclease activity (Calbiochem), was used at 45 μM. Cells were accumulated in mitosis upon Nocodazole (Sigma-Aldrich) treatment for 24 hours at 0.5 μg/μl. To label parental ssDNA, 5′-Iodo-2′deoxyuridine (IdU- Sigma-Aldrich) was used for 20 hours at 100 μM. To label DNA fibers: 5′-Chloro-2′-deoxyuridine (CldU- Sigma-Aldrich) was used at 50 μM, IdU was used at 250 μM for 20 min.

For DNA fiber assay, A549 cells were transiently transfected with SCAT7 targeting GapmeR along with negative control as previously described. Forty-eight hours post transfection, cells were subjected to two consecutives 30 min pulses with 0.1 mM 5-Chloro-2'-Deoxyuridine (CldU) and 0.1 mM 5-Iodo-2'-Deoxyuridine (IdU) (Sigma Aldrich) followed by three cold PBS washes. Cells were harvested and counted. 50 × 10⁵ cells/sample were used for gel plugs preparation using the FiberPrep DNA extraction kit (Genomic Vision, Begneux, France) and Gel plug molds (Bio-Rad, Hercules, CA, USA) following the manufacturer's protocol. DNA samples in agarose plugs were sent to Genomic Vision facility (Bagneux, France) for DNA stretching into coverslips and data acquisition on the FiberVision platform (Genomic Vision). We performed DNA fiber analysis using the software FiberStudio version 0.9.12 (Genomic Vision).

Cells were labeled with CldU (5-chloro-2-deoxyuridine, Sigma-Aldrich) for 20 min, washed twice with pre-warmed PBS before labeling cells with IdU (5-iodo-2-deoxyuridine, Sigma-Aldrich) in ten times higher concentration for another 20 min. During the IdU pulse, cells were either treated with MMC (3 μM) or H₂O as control. For TRC-experiments cells were pre-treated with DRB (20 μM) or its solvent DMSO 60 min prior to start of CldU-labeling and throughout the whole CldU- as well as IdU-labeling. Subsequently, cells were washed, harvested and resuspended in PBS. 2500 cells were transferred to a slide, lysed with 6 μl of 0.5% SDS, 200 mM Tris–HCl, pH7.4, 50 mM EDTA, and incubated at room temperature for 6 min. Slides were tilted about 20° to allow DNA to spread via gravity, covered with aluminum foil, air-dried for 7 min, fixed for 5 min with 3:1 methanol:acetic acid (prepared fresh), air dried for 7 min, and stored in 70% ethanol at 4°C overnight or directly afterwards processed for denaturation/deproteination in 2.5 N HCl for 1 h, followed by Immunofluorescence staining