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. 2017 Nov 20;6:e29303. doi: 10.7554/eLife.29303

Figure 3. MYPT1 limits kinetochore Plk1 localization and phosphorylation.

(A) U2OS before (CT) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) stained for DNA, centromere-specific human antisera (ACA), tubulin, and Plk1 as indicated. Scale bar, 5 µm. (B) Quantification of the intensity of kinetochore Plk1 staining in U2OS before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in prometaphase (Prometa) and metaphase (Meta) cells (n ≥ 100 kinetochores per condition. p<0.0001, unpaired, two-tailed t-test). (C) U2OS before (CT) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in either prometaphase (Prometa) or metaphase (Meta) stained for DNA, centromere-specific human antisera (ACA), tubulin, and pPlk1-Thr210 as indicated. Scale bar, 5 µm. (D) Quantification of the intensity of kinetochore pPlk1-Thr210 staining in U2OS cells before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in prometaphase (Prometa) and metaphase (Meta) cells (n ≥ 100 kinetochores per condition. p<0.0001, MYPT1KD prometaphase vs metaphase, p<0.0001, CT vs MYPT1 KD prometaphase; p=0.01 CT vs MYPT1 KD metaphase; unpaired, two-tailed t-test).

Figure 3.

Figure 3—figure supplement 1. Whole cell lysates compared for changes in protein expression.

Figure 3—figure supplement 1.

Western blots using whole cell lysates from U2OS before (Control) and after si-RNA-mediated MYPT1 knockdown (MYPT1 KD) for the indicated protein targets. Numbers indicate protein levels relative to actin loading control in each column.
Figure 3—figure supplement 2. Analysis of Plk1 levels.

Figure 3—figure supplement 2.

U2OS before (CT) and after si-RNA-mediated Cyclin A-knockdown (CycA KD) stained for DNA, centromere-specific human antisera (ACA), and Plk1 as indicated. Scale bar, 5 µm. Quantified intensity levels. (n ≥ 100 kinetochores/condition. p<0.0001 CT vs CycA KD prometaphase cells; p=0.0012 CT vs CycAKD metaphase cells).
Figure 3—figure supplement 3. Analysis of pPlk1 levels.

Figure 3—figure supplement 3.

Quantification of the intensity of kinetochore pPlk1-Thr210 staining in U2OS cells before (Control) and after si-RNA-mediated Cyclin A knockdown (Cyc A KD) in prometaphase (Prometa) and metaphase (Meta) cells (n ≥ 100 kinetochores/condition. p<0.0001 for CycA KD prometaphase vs metaphase, p<0.0001 for CT metaphase vs CycA KD prometaphase; p=0.001 for CT vs CycAKD metaphase).