PP6 and Aurora A regulate the size of the mitotic spindle. (A) Metaphase spindle size (mean ± SD; n = 12–13) in parental and PPP6C KO HeLa cell lines stained for active Aurora A pT288, tubulin, and DNA. Statistical significance was analyzed using an unpaired two-tailed t test with Welch’s correction (***, P < 0.001). (B) Metaphase spindle size (mean ± SD; n = 15–29) in parental and PPP6C KO HeLa cell lines after 30 min treatment in the presence (+) or absence (−) of Aurora A inhibitor (AurA-i). Statistical significance was analyzed using a Brown-Forsythe ANOVA (**, P < 0.01; ***, P < 0.001; ****, P < 0.0001). (C) Parental and PPP6C KO cell lines were treated with STLC for 3 h to arrest cells in mitosis with monopolar spindles and then treated for 30 min in the absence (Control) and presence of Aurora A (AurA-i) or Aurora B (AurB-i) inhibitors. The cells were then stained for DNA and CENP-C. Monoastral spindle diameter (mean ± SD; n = 9–21) is shown for the different conditions. Statistical significance was analyzed using a Brown-Forsythe ANOVA (****, P < 0.0001). (D) Time-lapse imaging of DNA segregation in parental and PPP6C KO cells. NEBD was taken as the start of mitosis. Anaphase is shown with higher time resolution with arrows to mark anaphase spindle defects in PPP6C KO cells. Arrows indicate chromosomes escaping the anaphase spindle. (E and F) Mitotic progression from NEBD to anaphase onset (E; the line marks the median value) and cumulative mitotic index in parental and PPP6C KO cells (F; n = 26–28). PPP6C KO cells show extended mitosis and delayed mitotic exit. (G) Mitotic chromosome spreads from parental and PPP6C KO HeLa cell lines. Arrows indicate broken or unpaired chromosomes. (H) Flow cytometry was used to measure cell cycle distribution (mean ± SD; n = 3) and ploidy of parental and PPP6C KO HeLa cells. Plots show counts of DNA content with dotted lines to mark 2c and 4c in the parental control cell line.