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. 2016 Nov 1;129(21):4130–4142. doi: 10.1242/jcs.190215

Fig. 4.

Fig. 4.

PRL-3 accelerates cytokinesis. (A) Representative images of metaphase cells in 72-h parental MDCK cysts or cysts that stably expressed GFP–PRL-3-WT or GFP–PRL-3-C104S (green). Blue, nuclei; red, γ-tubulin. Arrows indicate the orientation of the mitotic spindles. The spindle angle was analyzed. deg, degree. MDCK and PRL-3-C104S, n=21; PRL-3-WT, n=22. Scale bars: 10 µm. (B) Representative images of cells undergoing cytokinesis in 72-h parental MDCK cysts or cysts that stably expressed GFP–PRL-3-WT or GFP–PRL-3-C104S (green). Blue, nuclei; red, actin; magenta, anillin (cleavage furrow). Dashed boxes highlight the cell undergoing abscission. Merged and split channels of the selected region are shown below the picture. Asymmetry was measured when the cleavage furrow was around 50% ingressed (measured as distance between apical and basal membrane). A line tangent to the apical membrane and another tangent to the basal membrane were drawn. The distance between each line and the cleavage furrow was measured to determine the ingression for both the basal and the apical side, and the ratio between the basal and apical membrane ingression was plotted. Symmetric abscission=1. n=45. Scale bars: 10 µm. (C,D) 48-h MDCK cysts overexpressing GFP–PRL-3-WT (C) or GFP–PRL-3-C104S (D). Arrows indicate PRL-3 accumulation in the ingressed furrow. n=30. See also Movie 5. Scale bars: 10 µm. (E–H) Cell cycle analysis. MDCK cells (E), MDCK cells treated with 2 μM of ZM447439 (Aurora kinase B inhibitor) over 48 h (F) and MDCK cells that stably expressed GFP–PRL-3-WT (G) or GFP–PRL-3-C104S (H) were analyzed by performing flow cytometry. Blue, G0 and/or G1; olive green, S phase; green, G2; white, tetraploid cell. (I) The time taken for cells to undergo abscission was measured in MDCK cysts and MDCK cysts expressing GFP–PRL-3-WT or GFP–PRL-3-C104S that had also been transfected with Aurora kinase B to track cytokinesis over time. See also Movies 6 and 7. n=50. Median (A) or mean (B,I) values for each condition from three biological replicates is shown as a line. ns, not significant; ***P≤0.001; ****P≤0.0001 (A,B,I, ordinary two-tailed one-way ANOVA with Tukey's multiple comparison test).