EB3 is required for stabilization of the midbody upon mitotic exit. (A) HeLa cells were filmed in phase contrast to determine the number of failed cytokinesis events. Yellow arrowheads indicate the first time-frame where the midbody could be observed. The broken lines indicate the region that was used to generate the kymograph of the midbody position. Horizontal bar, 10 µm; vertical bar, 50 min. Note the formation of a binucleated cell in an EB3-depleted cell, where the midbody fails to stabilize. In the low-magnification images, white arrows indicate the cells that were tracked in this particular example. 0 min corresponds to the first frame in anaphase. (B) Quantification of the number of failed cytokinesis per total divisions observed upon EB3 and/or EB1 RNAi and respective rescue efficiencies with ectopic constructs. Error bars indicate mean ± SEM. *, P < 0.05 when compared to control RNAi using nonparametric ANOVA followed by a post-hoc Dunn’s test. (C) Quantification of midbody stability in control RNAi and EB3-depleted cells. Midbody movement amplitude and frequency were extracted from kymographs generated in the midbody region. Movement amplitude corresponds to the lateral deviation of the midbody throughout time. Movement frequency was defined as the number of midbody oscillations per unit of time. (*, P < 0.001 using nonparametric ANOVA followed by a post-hoc Dunn’s test). All experiments were done in triplicate, and n represents the number of cells quantified in each condition. (D) FRAP analysis of the midbody region in cells expressing α-tubulin–GFP. The percentages of fluorescence recovery and half-time recovery were determined by applying a double (control and EB1 RNAi) or single (EB3 RNAi) exponential fitting to the recovery curve. Time is given in minutes:seconds. Horizontal bar, 10 µm; vertical bars, 3 µm. *, P < 0.05 when compared to control RNAi using nonparametric ANOVA followed by a post-hoc Dunn’s test.