Anti idu

24 h after siRNA transfection, U-2-OS cells were labeled for 10 min with 10 µM IdU (Sigma-Aldrich) followed by 20-min labeling with 100 µM CldU (MP Biomedicals). 2 µl of cells resuspended in ice-cold PBS was deposited on a microscope slide and incubated with 7 µl of spreading buffer (200 mM Tris-HCl, pH 7.5, 0.5% SDS, and 50 mM EDTA) for 3 min. The slides were tilted 15° to stretch the DNA fibers (Bianco et al., 2012 (link)). 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 (anti-CldU [AbCys SA], anti-IdU [BD], and anti-ssDNA [EMD Millipore]) and corresponding secondary antibodies conjugated with Alexa Fluor 488, 546, or 647 (all obtained from Invitrogen). Statistical analysis was performed using Prism 5 (GraphPad Software).

Mouse monoclonal antibodies against FEN1, CDK2, and CIdU were from Abcam. Anti-IdU was from BD Biosciences; anti-FLAG was from Santa Cruz Biotechnology; antibodies against PCNA, FEN1, SUMO-1, HUS1, RAD9, RAD1, and β-actin were from Proteintech; anti-γH2AX and secondary antibodies were from Cell Signaling Technology. Mouse/rabbit Duolink^® PLA and anti-FLAG M2 magnetic beads were from Sigma-Aldrich. Dulbecco’s modified Eagle’s medium (DMEM) with high glucose, fetal bovine serum (FBS), penicillin and streptomycin (PS), and phosphate buffer solution (PBS) were obtained from HyClone, GE Healthcare.

Cells were pulse-labeled with 25 μmol/L ldU (Sigma-Aldrich; no. I7125) for the first 30 minutes, followed by 250 μmol/L CIdU (Sigma-Aldrich; no. C6891) for 30 minutes. The cells were trypsinized and resuspended in PBS then diluted to the concentration of 1.0 × 10⁵ to 1.0 × 10⁶ cells/mL. At the end of an APS-coated glass slide (Matsunami; no. SUAPS1190), 2 μL of cell suspension was placed. After air-drying for 8 minutes, 7 μL of fiber lysis solution (200 mmol/L Tris-HCl [pH 7.5], 50 mmol/L EDTA, 0.5% SDS) was pipetted on top of the cell suspension and mixed gently. Cell lysis proceeded for 5 minutes. The slides were tilted at 15° to allow the DNA to spread down to the bottom of the slide. Slides were air-dried for 15 minutes and fixed in methanol/acetic acid (3:1). After washing with distilled water, DNA was denatured in 2.5 M HCl for 80 minutes. The slides were washed with PBS three times and blocked in 5% BSA in PBS for 1 hour. After blocking, the slides were incubated with primary antibodies, anti-IdU (BD; no. 347580), and anti-CldU (Abcam; no. ab6326) and followed by secondary antibodies, donkey anti-mouse IgG (H+L) conjugated with Alexa Fluor 488 (Thermo Fisher Scientific; no. A32766), and donkey anti-rat IgG (H+L) conjugated with Alexa Fluor 594 (Thermo Fisher Scientific; no. A21209).

Cells were labeled with 50 μM CldU (Sigma), exposed to HU (4 mM) and labeled with 50 μM IdU (Sigma) as indicated in the figures. DNA fibers were spread as previously described (35 (link)), and fiber tracts were detected using anti-IdU (BD Biosciences, 347580) and anti-CldU (Novus Biologicals, NB500-169) primary antibodies and Alexa Fluor 488 and 555 secondary antibodies (Invitrogen). Fibers were imaged at 60× magnification with oil immersion using a Zeiss microscope and analyzed with ImageJ.