Laser micro-irradiation analysis was performed as previously described with a few modifications [45 (link),46 (link)]. We seeded 2 × 105 of cultured cells expressing wild-type olNbs1-Venus or olNbs1 (H170)-Venus into the glass-bottom dishes and cultured the cells for 2 days at 33°C. After heat treatment (2 h) at 41°C to induce olNbs1-Venus or olNbs1 (H170)-Venus expression, the cells were incubated overnight and then incubated with 4 μM Hoechst 33258 (Wako, Osaka, Japan) in L15 medium supplemented with 20% FBS for 10 min at 33°C. Then, the cells were washed in L15 medium (phenol red free) before laser irradiation. Images were obtained using a SP6 confocal microscope (Leica Microsystems, Wetzlar, Germany) and analyzed using LAS AF software (Leica Microsystems). We set a strip-shaped region of interest (ROI) and 5 shots of 405 nm laser was irradiated into the ROI, inducing DSBs in the restricted part of the nuclei. Seventeen images were captured at 15 s intervals from 1.3 s to 241.3 s after the micro-irradiation, and then 3 more images were captured at 30 s intervals. The mean fluorescence intensity of the irradiated ROI in the nucleus was digitalized using Image J. Fold increase (F. I.) at each time point was calculated by following equation; F. I. (%) = 100 × ((mean intensity at each time point)–(mean intensity at t = 1.3 s))/(mean intensity at t = 1.3 s).
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