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. 2021 May 3;56(9):1253–1267.e10. doi: 10.1016/j.devcel.2021.04.005

Figure 4.

Figure 4

Depletion of both kinesin-6 MKLP1 and kinesin-6 MKLP2 with inactivation of EG5 induces a partial block in chromosome segregation by disrupting spindle elongation

(A) Live-cell images of control, MKLP1- and MKLP2-siRNA co-depleted and MKLP1- and MKLP2 siRNA co-depleted 40-μM STLC-treated RPE-1 cells stably expressing CENP-A-GFP and centrin1-GFP. kin, kinetochore; cen, centrosome. White arrowheads indicate centrioles. Horizontal gray bars in the kymographs indicate the onset of anaphase.

(B) Quantification of velocity of spindle elongation (n = 34, 17, 12, and 11 cells, from left to right). Three independent experiments for every condition regarding siRNAs or non-targeting treatments, while number of independent experiments regarding STLC treatment is equal to the number of cells. Black data dots in every treatment correspond to the measurements from the exemplar cells shown on the time-lapse images and kymographs (A). Statistics: t test (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

(C) Quantification of net spindle elongation from anaphase onset to 5-min afterward for treatments as indicated (n = 7, 10, 9, and 10 cells, from left to right). Time is shown as minutes. Images are the maximum projection of the acquired z stack. Time 0 represents anaphase onset. Horizontal scale bars, 1 μm. Vertical scale bars, 1 min.

(D) Immunofluorescence images of fixed RPE-1 cells depleted of target proteins by treatment with indicated siRNAs stably expressing CENP-A-GFP and centrin1-GFP (magenta) stained with AlexaFluor594 conjugated with antibody specific to the indicated target protein (green KIF4A and red MKLP2). Chromosomes were stained with 1 μg/mL DAPI solution (cyan). Scale bars, 2 μm (D).

(E) Quantification of mean signal intensity measured in the spindle midzone of KIF4A (top) and MKLP2 (bottom) antibodies conjugated with AlexaFluor594 versus spindle length in the indicated conditions (8 MKLP2 depleted and 5 control cells). Two independent experiments for every condition regarding siRNAs or non-targeting treatments. See also Figure S4.