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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Nat Cell Biol. 2011 Aug 28;13(10):1265–1271. doi: 10.1038/ncb2327

Figure 4.

Figure 4

B56-PP2A depletion increases the phosphorylation of Aurora B substrates and Aurora inhibition suppresses the B56-PP2A siRNA phenotype. (a–c) RPE1 cells were transfected with control or B56-PP2A siRNA (pool 2) and (a, c) incubated with nocodazole (3.2 µM, 60 min) before fixation, or (b) fixed, and stained using indicated antibodies. Images are maximum intensity projections with equivalent scaling. The signal at kinetochores was measured, processed, and normalized to the average value in control siRNA cells. Histograms show intensity distributions from one experiment. (a) KMN network targeting is preserved in B56-PP2A siRNA cells. In B56-PP2A siRNA cells, the mean kinetochore staining intensities were calculated for Dsn1 (0.85 ± 0.11), Knl1 (1.41 ± 0.23), and Hec1 (1.05 ± 0.08) relative to control cells (n=2–3 experiments, >75 kinetochores from 5 cells per condition, per time). (b–c) Analysis of Aurora B substrate phosphorylation. (b) In prometaphase cells, kinetochores with an inter-kinetochore stretch of 1.2 to 1.5 µM were analyzed. In B56-PP2A siRNA cells, the mean phosho-Dsn1 and phospho-Knl1 intensity was 1.79 ± 0.32 and 2.26 ± 0.07, respectively (n=2 experiments, >50 kinetochores from 5 cells per condition, per time). (c) In nocodazole-treated B56-PP2A siRNA cells, the mean intensity of phospho-Dsn1 and phospho-Knl1 was 1.03 ± 0.13 and 1.21 ± 0.15, respectively, relative to control cells (3 experiments, >60 kinetochores from 5 cells per condition per experiment). (d–e) RPE1 cells treated with control or either of two pools of B56-PP2A siRNA (1, 2) were incubated in MG132 (10 µm, 15 min), followed by addition of Hesperadin (50 nM) or ZM447439 (1 µM) or control solvent (DMSO) for 45 min. (d) The frequency of mitotic cells with few or no cold-stable K-fibers is plotted (n=3 experiments, >80 cells per condition per time). (e) Maximum intensity projection of tubulin (green) and an overlay with kinetochores (CREST, red) in B56-PP2A siRNA (pool 2) cells treated with indicated inhibitor. Insets are 2x enlargement of the optical sections spanning the boxed regions. Scale bars, 5 µm. Mean ± s.e.m provided.