Laser micro-irradiation and imaging
U2OS cells were cultured and transiently transfected with wild type and the 3 mutant CSB-PGBD3-RFP plasmids together with or without OGG1-GFP plasmid using Lipo2000 (Invitrogen). DNA damage was induced in the nuclei of cultured cells by micro-irradiation with a pulsed nitrogen laser (Photonics Instruments; 365 nm, 10 Hz pulse) [31]. Briefly, cells were seeded onto 35-mm glass bottom dishes (MatTek) overnight before being visualized with a Nikon Eclipse Ti-E inverted microscope equipped with a computer-controlled MicroPoint laser Ablation System (Photonics Instruments) for time-lapse imaging. The output of the laser power was set at 70–90% of the maximum as indicated by the manufacturer. During micro-irradiation and imaging, cells were maintained at 37°C. The growth medium was replaced by CO2-independent medium (Invitrogen) before analysis. The mean fluorescence intensity of CSB-PGBD3-RFP foci in the laser induced damage sites and that of the other nuclear area were determined after subtraction of the extranuclear background signal. The ratio of the foci fluorescence intensity over the other nuclear area fluorescence intensity was used to indicate the quantification of the recruitment. The average and SE of a total of at least 10 cells are depicted in the graph.