Depletion of EB proteins leads to different phenotypes during mitosis. HeLa cells expressing H2B-GFP/α-tubulin–mRFP were depleted of EB1 and/or EB3 and imaged by spinning disk microscopy. (A) Quantification of NEB-to-anaphase duration (*, P < 0.05 using nonparametric ANOVA followed by a post-hoc Dunn’s test). (B) Frames from time-lapse movies showing the phenotypes quantified. The arrow indicates a daughter cell that failed to attach to the substrate. Bars, 10 µm. (C) Cumulative quantification of mitotic phenotypes in all experimental groups. The spindle was considered “tilted” when the two poles were not on the same focal plane for >5 min. (D) HeLa cells were immunostained with an α-tubulin antibody, and top (xy) and lateral (yz) projections of the spindle were used to identify spindle poles and quantify spindle angle in relation to the substrate. Red circles highlight the spindle poles. Horizontal bar, 5 µm; vertical bar, 1.5 µm. (E) Quantification of spindle angles relative to the substrate in prometaphase (PM), metaphase (M), anaphase (A), and telophase/cytokinesis (T/C) for all treatment groups (*, P < 0.05 using parametric ANOVA followed by a post-hoc Student-Newman-Keuls test). Experiments were done in triplicate and N represents the number of cells quantified in each condition.