For fiber assays, 30 hours after siRNA transfection, U-2-OS cells were pulsed with 10 μM 5-iodo-2′-deoxyuridine (IdU) (Sigma-Aldrich) for 10 min, followed by 20-min labeling with 100 μM CldU (MP Biomedicals). Cells resuspended in 2 μl of ice-cold PBS were incubated with 7 μl of spreading buffer [200 mM tris-HCl (pH 7.5), 0.5% SDS, and 50 mM EDTA] for 3 min on a slide, and DNA fibers were stretched by tilting. After fixation with methanol/acetic acid (3:1), DNA was denatured with 2.5 M HCl and blocked (PBS with 1% BSA and 0.1% Triton X-100) before staining with primary antibodies. Single-molecule analysis of DNA replication by molecular combing was performed as described in protocol 36 available from the EpiGeneSys Network of Excellence website. In brief, after 30 hours of siRNA transfection, U-2-OS cells were labeled with 10 μM IdU (Sigma-Aldrich) for 10 min, followed by 20-min labeling with 100 μM CldU (MP Biomedicals). Cells were harvested immediately after the pulse and molded into low-melting agarose plugs. Plugs were treated with proteinase K buffer, melted at 67°C, and then digested by β-agarase. DNA was combed on silanized coverslips (Genomic Vision), denatured by 2.5 M HCl, and probed by the primary antibodies. Graphs show one representative experiment of two biological replicates, and lines represent medians. Images for both assays were collected using a DeltaVision system (Applied Precision) with a UApo/340 40×/1.35 NA oil objective lens, and the length of CldU-labeled tracks was measured with SoftWoRx 5.0.0 (Applied Precision).

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