Behavior of Mad2 during mitosis in RPE1 cells. (A–C) Selected time points from multimode 3D time-lapse recordings of cells expressing Mad2-Venus. Single focal planes from DIC volumes (top) and maximum-intensity projections of the entire cell (bottom) are shown for each time point (time in minutes from NEB). (A) The cell is treated with 3 µM nocodazole to prevent formation of spindle microtubules. (B) The cell is under normal culture conditions. Arrow denotes a single Mad2-positive kinetochore that transiently appears ∼4 min before anaphase onset. (C) The cell is treated with 20 nM GSK923295 to inhibit CenpE. Arrows point at the perpetually monooriented chromosomes whose kinetochores recruit large amounts of Mad2 at later stages of mitosis. (A′–C′) Amounts of Mad2 recruited to the kinetochores under the same conditions as in A–C. Each time point is characterized by the median (red marks), range of 25th–75th percentiles (box), full range of the data points (whiskers), and outliers deviating by >2.698 σ from the mean (red crosses). (D) Heatmaps presenting distribution of kinetochores with various amounts of Mad2. Lookup tables are normalized to the bins with maximal number of kinetochores in each experimental condition. (E) The number of Mad2-positive kinetochores per cell under various conditions. Error bars represent SD.