ETAA1 activation of ATR is required for proper chromosome alignment and a fully functional SAC. (A and B) HCT116 WT, ETAA1ΔAAD, and TOPBP1-AID cells expressing GFP-H2B for chromatin visualization were examined by live cell imaging and scored for defects during mitosis. ATRi or IAA was added 1.5 h before beginning imaging. Representative time-lapse images are shown in A. White arrows indicate anaphase bridges and misaligned or lagging chromosomes. (B) Quantification of mitotic defects from live cell imaging experiments. Total number of cells examined for each cell type from three independent experiments is indicated in parentheses. (C) Representative immunofluorescence images of normal and misaligned chromosomes during metaphase. (D) HCT116 WT, ETAA1ΔAAD, and TOPBP1-AID cells were arrested for 16 h with CDK1i, treated with IAA or ATRi for 2 h, released from CDK1i, and fixed. Metaphase cells were scored for the number of misaligned chromosomes. (E) U2OS WT, ΔETAA1, and ΔETAA1 cells stably expressing ETAA1 were examined for chromosome misalignment as in D. D and E display the mean and standard deviation of three independent experiments in which at least 100 metaphases were examined per condition. (F) Synchronized HCT116 cells expressing H2B-GFP were released from a double thymidine block, and taxol, ATRi, or CHK1i were added 1.5 h before starting imaging. (G) Same as in F, but HCT116 WT and two clones of ETAA1ΔAAD and TOPBP1-AID cells were examined for ability to sustain mitotic arrest during taxol treatment. (F and G) The total number of cells examined for each cell type from three independent experiments is listed in parentheses. Bars: 5 µm (A and C).