Fig 1: PPP6C KO cells show elevated Aurora A dependent phosphorylation of the kinetochore protein NDC80. (A) Parental and PPP6C KO cells depleted of NDC80 were stained for DNA, tubulin, and the nuclear pore marker NUP153, and imaged at 40× magnification to visualize nuclear morphology. Arrows mark micronuclei in NDC80-depleted parental cells. (B) Mitotic lysates of parental and PPP6C KO HeLa cells were blotted for the proteins listed in the figure. Overall NDC80 phosphorylation was monitored using a Phos-tag gel. The relative levels of NDC80 S55 phosphorylation, active Aurora A (pT288), and active Aurora B (pT232) were measured (mean ± SEM; n = 4–8). Statistical significance was analyzed using an unpaired two-tailed t test with Welch’s correction (***, P < 0.001; ****, P < 0.0001). To control for antibody phospho-selectivity, HeLa cell lysates were mock- (-) or ?-phosphatase–treated (+?-PPase) and blotted for NDC80, NDC80 pS55, and pS62. (C) Dephosphorylation kinetics of Aurora A pT288 and NDC80 pS55 were followed in extracts of parental and PPP6C KO cells. Graphs show dephosphorylation kinetics (mean ± SD; n = 3). (D) Mitotic lysates of parental and PPP6C KO HeLa cells treated for 10 min with Aurora A (AurA-i) and two different Aurora B (AurB-i) kinase inhibitors in the combinations shown were blotted for the proteins listed in the figure. To increase sensitivity, NDC80 was isolated by immunoprecipitation for NDC80 pS55 blots. An extended analysis of this experiment, including blots for additional NDC80 phosphorylation sites, is shown in Fig. S3 D. (E) Parental and PPP6C KO HeLa cells in different kinase inhibited and control conditions from D stained for NDC80 pS55, NDC80, and DNA. (F) HeLa cells were depleted of endogenous TPX2 for 72 h or treated with a non-targeting control siRNA (siControl). Cells were fixed and then stained for TPX2, CENP-C, DNA, or antibodies to specific NDC80 pS55. (G and H) NDC80 pS55, pS44, and pS62 signal at kinetochores (G) and monoastral spindle size in siControl and siTPX2 (H; mean ± SD; n = 5–29). Statistical significance was analyzed using an unpaired two-tailed t test with Welch’s correction (**, P < 0.01; ****, P < 0.0001). (I) HeLa cells were depleted of Aurora A or Aurora B for 72 h using siRNA or treated with a non-targeting control siRNA (siControl). Cells were fixed and then stained with antibodies to specific NDC80 phosphorylation sites. Images for NDC80 pS55 are shown in the figure, pS44 and pS62 are shown in Fig. S4 D. (J and K) NDC80 pS55, pS44, and pS62 signal at kinetochores expressed relative to NDC80 (J) and monoastral spindle size in siControl, siAurora A, and siAurora B treated cells (K; mean ± SD; n = 5–24). Statistical significance was analyzed using a Brown-Forsythe ANOVA (**, P < 0.01; ****, P < 0.0001). Source data are available for this figure: SourceData F4.