Fig. 2.

AURKA destruction is not required for pT288-AURKA down-regulation at mitotic exit. (A,B) There is no mitotic exit destruction of AURKA in FZR1 KO cells (FZR1^KO). AURKA–Venus (A) or H2B–GFP (B) were transiently transfected into both U2OS and U2OS FZR1^KO cells. In A, quantifications of total fluorescence measurements from single mitotic cells were used to generate degradation curves for AURKA–Venus. Fluorescence values for individual curves were normalized to the last frame before anaphase onset, and all curves were in silico synchronized to anaphase. n=10 cells. Data are mean±s.d. In B, H2B–GFP fluorescence was used to score DNA as condensed or decondensed in cells undergoing mitotic exit (example shown in lower panels). Percentage of cells with condensed DNA over time was plotted as a measure of cumulative mitotic exit. n≥10 cells. (C–E) AURKA activity scored by pT288 is not affected by FZR1^KO during mitotic exit. For the western blots in C, U2OS and FZR1^KO cells were synchronized to prometaphase using 5 μM STLC and released by checkpoint inhibition using 10 μM AZ3146, with extracts harvested at the times indicated. Lysates were analysed by immunblot with antibodies against AURKA, pT288-AURKA and other mitotic regulators and are representative of three independent experiments. D and E show AURKA and pT288-AURKA staining associated with individual centrosomes/spindle poles in metaphase (M) versus telophase (T) cells (left-hand panels). Fluorescence values were measured as in Fig. 1 and are presented as scatter plots, with mean±s.d. indicated, for total AURKA (D) and pT288-AURKA (E) in both U2OS and FZR1^KO. All values were normalized to the mean value from control metaphase cells. ns, not significant; **P<0.001; ***P<0.0001 (Student's t-test). D, n≥11 from one experiment; E, n≥23 from two experiments. Scale bars: 10 μm.