The IAA-induced cell cycle arrest is not caused by a prolonged mitosis. (A) Representative images of anaphase cells at indicated times after IAA addition. Cells were costained with Centrin and CEP192. Bars: (main) 5 µm; (inset) 0.5 µm. (B) Quantification of the percentage of cell divisions occurring with the indicated number of centrioles at each spindle pole. Measurements were obtained from fixed samples at the indicated times after IAA addition. Bars represent the mean of >60 cells from two independent experiments. (C) Schematic showing the expected dynamics of centriole dilution after IAA treatment. Cells that undergo mitosis within 6 h of IAA addition almost always contain four centrioles, which dilute out as shown in subsequent divisions. (D) Graph showing the prometaphase duration and proliferative capacity of IAA-treated Plk4^AID/AID cells. Each bar represents a daughter cell, its height represents the prometaphase duration of the mother cell, and its color represents the fate of the daughter. Only cells that underwent their first mitosis within 6 h of IAA treatment were analyzed. The dashed red line indicates the maximum time that IAA-treated cells spend in prometaphase before undergoing a cell cycle arrest (Fig. S3 D). Data are taken from two independent experiments (n = 199 prometaphases). (E) Immunoblot showing the levels of phosphorylated LATS, YAP, and Histone H2AX in IAA-treated Plk4^AID/AID and parental RPE1 cells. Doxorubicin treatment was used as a control to induce DNA damage. (F) Graph showing the fold increase in cell number in IAA-treated Plk4^AID/AID cells grown in normal (21%) or low (3%) oxygen conditions. Points show the mean of two independent experiments performed in triplicate. All error bars in the figure represent the SEM.