Actinomycin D treatment
For actinomycin D treatment, HeLa-EGFP-hOGG1 cells were grown on chamber slides and synchronized at the G1/S boundary by two cycles of thymidine blockage. Cells were allowed to enter S-phase and actinomycin D was added 1 h after block release and incubated for 4 and 5 h, respectively. Cells were then washed twice with phosphate-buffered saline (PBS), fixed with 96% ethanol (anti-nucleolin) for 10 min at room temperature or 4% PFA (anti-fibrillarin and anti-RPA135) for 20 min and permeabilized with 0.5% Triton X-100 for 5 min at 4°C and washed again with PBS. Cells were air-dried before incubation with primary and secondary antibodies diluted in PBS with 1% BSA. As primary antibody monoclonal anti-nucleolin (clone 3G4B2; Upstate Biotechnology), RPA40 (N-20) (sc-17700; Santa Cruz), RPA135 (N-17) (sc-17913; Santa Cruz) and anti-fibrillarin (AFB01, cytoskeleton) were used and incubated overnight at room temperature. Incubation with secondary antibody (Alexa 594, 590/617; Molecular Probes) was carried out for 30 min at room temperature. DNA was stained with DAPI for 10 min at room temperature. Slides were mounted using the DAKO fluorescence mounting medium and examined using a Zeiss Axioplan 2 fluorescence microscope. Images were obtained using CoolSNAP (Photometrics) and then processed for publication using Jasc Pain Shop Pro (Jasc software).