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

Figure 4. MYPT1 promotes efficient error correction by regulating Plk1.

(A) Microtubule turnover rates were measured and k-MT half-life was calculated from RPE1 cells before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) in prometaphase and metaphase cells as indicated (n ≥ 10 cells/condition from ≥2 independent experiments. ***Indicates p<0.0001, **indicates p=0.0096, unpaired, two-tailed t-test. (B) Microtubule turnover rates were measured and k-MT half-life was calculated from U2OS cells before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) and with the addition of the Plk1-specific inhibitor Bi-2536 in prometaphase and metaphase cells as indicated (n ≥ 15 cells/condition from ≥2 independent experiments. CT cells p<0.0001; MYPT1 KD cells p=0.04; MYPT1KD +BI-2536 p=0.0038; unpaired, two-tailed t-test). (C) Microtubule turnover rates were measured and k-MT half-life was calculated from U2OS cells before (Control) and after si-RNA-mediated Cyclin A2-knockdown (CycAKD) or transfection with plasmid containing either full-length MYPT1 (MYPT1), phosphonull (MYPT1-473A), phosphomimetic (MYPT1-473D) or tandem phosphomimetic (MYPT1-472:473DD) in prometaphase and metaphase cells as indicated (n ≥ 10 cells/condition in all conditions except MYPT1-473A metaphase where n = 9 cells). ***Indicates p<0.0001, **indicates p=0.004, unpaired, two-tailed t-test. (D) Fraction of anaphase cells displaying lagging chromosomes in RPE1 (Top) and USOS (Bottom) cells before (CT) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) (n ≥ 300 anaphases/condition. p-values n.s., unpaired two-tailed t-test). (E) Fraction of anaphase cells displaying lagging chromosomes following release from monastrol treatment in U2OS cells before (Control) and after si-RNA-mediated MYPT1-knockdown (MYPT1 KD) (n ≥ 800 anaphases/condition from two independent experiments. p<0.0001, Chi square contingency analysis).

Figure 4.

Figure 4—figure supplement 1. Fluorescence Dissipation After Photoactivation (FDAPA) Representative Curves From Microtubule Stability Measurements.

Figure 4—figure supplement 1.

(Top) Examples of normalized fluorescence dissipation after photoactivation (FDAPA) in prometaphase for untreated (Control) and MYPT1-KD single cells that are representative of the mean. These data are then used to determine the rate of fluorescence dissipation over time, from which a microtubule half-life is calculated. (Bottom) Examples of normalized fluorescence dissipation after photoactivation (FDAPA) in metaphase for untreated (Control) and MYPT1-KD single cells that are representative of the mean. These data are then used to determine the rate of fluorescence dissipation over time, from which a microtubule half-life is calculated.
Figure 4—figure supplement 2. Calcium Stabilization Assay.

Figure 4—figure supplement 2.

(Top) Representative images of assay quantified in (D). Scale bar, 5 µm. (Bottom) Quantification of intensity of spindle tubulin staining in U2OS cells treated with calcium stabilization assay without (CT) and with si-RNA mediated MYPT1 KD in prometaphase or metaphase. n ≥ 20 cells/condition.
Figure 4—figure supplement 3. Quantifications of Other Mechanical Effects of MYPT1 in Mitotic Cells.

Figure 4—figure supplement 3.

Table listing quantification of spindle length and inter-kinetochore distance ±SEM. For spindle length, n ≥ 13 cells per condition as listed; for inter-kinetochore distance, n ≥ 100 kinetochore pairs per condition as indicated.
Figure 4—figure supplement 4. Analysis of Whole Cell Levels of Expression of Various MYPT1 Plasmids.

Figure 4—figure supplement 4.

Western blots using whole-cell lysates from U2OS cells before (CT) and after lipofectamine-mediated transfection of either full-length MYPT1, MYPT1-472:473DD, MYPT1-473A, or MYPT1-473D mutant plasmids, for MYPT1 and loading controls (Tubulin and Actin).