Real-Time Recruitment of DNA Repair Factors

GFP-NBS1, GFP-EXO1, GFP-Ku70, GFP-XLF, or GFP-XRCC4 were transfected into HCT116 DNA-PK_cs +/+, −/−, or KD/− cells with JetPrime^® (Polyplus) following the manufacturer's instructions. Twenty-four hours after the transfection laser micro-irradiation and real-time recruitment was performed with a Carl Zeiss Axiovert 200M microscope with a Plan-Apochromat 63X/NA 1.40 oil immersion objective (Carl Zeiss) as previously described (28 (link)). DSBs were generated with a 365-nm pulsed nitrogen laser (Spectra-Physics), which was directly coupled to the epifluorescence path of the microscope (28 (link)). Time-lapse images were taken via a Carl Zeiss AxioCam HRm camera. The cells were maintained in a CO₂-independent medium (Invitrogen) at 37°C during micro-irradiation and time-lapse imaging. Fluorescence intensities of the micro-irradiated area and control area were determined by Carl Zeiss Axiovision software, v4.5, and the intensity of irradiated was normalized to non-irradiated control area as previously described (26 (link)).