Figure 5. Abnormal spindle formation in PPP6C-depleted cells. (A) HeLa cells stably expressing EGFP-tagged histone H2B were transfected with control or PPP6C si08 duplexes for 48 h and then imaged. An image stack of 25 planes spaced 0.7 µm apart was taken at four stage positions every minute for 10–12 h. Exposure times were 10 ms for EGFP-tagged histone H2B using 1% of full lamp intensity. A brightfield reference image stack was also collected using 10-ms exposures. (B) HeLa cells stably expressing mCherry-tagged histone H2B and EGFP-tagged -tubulin were transfected for 48 h with control or PPP6C si08 duplexes and then imaged every minute at three stage positions for 10 h using a spinning-disk confocal microscope (Ultraview Vox). Laser set- tings were 3% with 30-ms exposure per channel, and 35 optical sections spaced 0.5 µm apart were taken for both channels. Representative examples of cells passing through mitosis are shown. The times are given in hours and minutes. Arrowheads mark abnormal spindle poles and lagging chromosomes. (C) The time taken to form a spindle and pass through mitosis was measured from the live-cell imaging and is plotted in the graphs (40 cells from three experiments). A histogram of spindle formation is also shown with a graph showing the observed defects in chromosome alignment and spindle poles (38 cells from three experiments). (D) HeLa cells were transfected with control or PPP6C si08 duplexes for 48 h. During the last 18 h, they were arrested with 2 mM thymidine. After washout of the thymidine, samples were collected at the times indicated, split, and used for FACS analysis or Western blotting. Mitotic index calculated from the FACS analysis is shown below the Western blot at the corresponding time point. The graph shows the ratio of phosphorylated/ total Aurora A at each time point (n = 2). Where present, the red and black lines indicate the phosphorylated and nonphosphorylated forms of Aurora A. Molecular mass is given in kilodaltons. Error bars indicate the standard error of the mean. Bars, 10 µm. Protein phosphates 6 regulates Aurora A • Zeng et al. 1321