Both microtubule polymerization and microtubule nucleation were promoted during DMSR. (A) GFP-EB3 comet tracks were recorded in RPE-1 cells under indicated treatments. Images of projections at 1 s (single frame), spanning 0–15 s (overlay), and representative EB3 comet tracks spanning 15 s with different colors are shown. Comets tracks were generated with KymographClear 2.0 and KymographDirect 2.1 in ImageJ software (see also Videos 1, 2, and 3). Scale bar, 20 µm. (B) Left: Representative kymograph images for the measurement of EB3 comet velocity. Right: Box plot shows quantitative analysis of GFP-EB3 comet velocities in live G1 phase GFP-EB3–expressing RPE-1 cells under different treatments. Center line, median; box limits, 25th and 75th percentile; whiskers, fifth and 95th percentile (see also Materials and methods). (C) Centrosome-dependent microtubule nucleation capacity was determined by live-imaging time-lapse experiments in G1 RPE-1 cells. Left: The first single frame of GFP-EB3 in steady status was shown. Right: Overlaid images showed increased centrosomal GFP-EB3 signals after IR. Arrowheads indicate centrosomes where GFP-EB3 signal was started (see also Videos 4, 5, 6, and 7). Scale bar, 20 µm. (D) Quantitative analysis of relative centrosomal GFP-EB3 intensity in overlaid images as in C was shown in box plot (n > 30). (E) Live-imaging time-lapse experiments of GFP-EB3 were performed in G1 RPE-1 cells 4 h after bleomycin treatment. Arrowheads indicate centrosomes where GFP-EB3 signal was started (see also Videos 8 and 9). Scale bar, 20 µm. ***, P < 0.001; **, P < 0.01. A.U., arbitrary units; UT, untreated.