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FIGURE 1:. Spindle assembly is compromised in mitotic cells that undergo MT regrowth. (A) Experimental design. Centrosome MTs are in red, chromatin dependent MTs are in green. (B) Images from time-lapse movies of H2B-eGFP/α-tubulin-mRFP HeLa cells undergoing mitosis after nocodazole washout. Top, control cell (MT Regrowth before mitosis). The three bottom rows show arrested mitotic cells going through mitosis after nocodazole washout (MT Regrowth during mitosis). In the two bottom rows, the cells enter anaphase after >5 h with a tripolar and a disorganized spindle. Tubulin is in green and H2B in red. Time is in hours:minutes. Time 0 corresponds to NEBD for the control cell and to nocodazole washout for MT Regrowth cells. Scale bar, 10 μm. (C) Time required for H2B-eGFP/α-tubulin-mRFP HeLa cells to enter anaphase after nocodazole washout. For control cells (MT Regrowth before mitosis), the values correspond to the time from NEBD to anaphase entry. For mitotic cells undergoing MT regrowth (MT Regrowth during mitosis), the values correspond to the time from nocodazole washout to anaphase entry. Box-and-whiskers plot. A total of 185 control cells and 149 MT regrowth cells were analyzed in four independent experiments. ***p < 0.001. (D) Spindle organization at anaphase onset. The proportion of bipolar (light blue), multipolar (gray), and disorganized (red) spindles was quantified for the cells analyzed in C. Mitotic cells undergoing MT regrowth (MT Regrowth) assemble significantly more multipolar and disorganized spindles than control cells (Control; p < 0.05). (E) Spindle organization in mitotic HeLa cells (Control, blue) or HeLa cells undergoing MT regrowth (MT Regrowth, red). Proportion of different spindle organizations 30 min after nocodazole washout or in control cells. MT regrowth cells have significantly fewer bipolar spindles properly oriented and a higher number of tilted and multipolar spindles than control cells. Representative IF images of different spindle organizations are shown below the graph. Data from 847 controls cells and 864 MT regrowth cells from four independent experiments, counting in each at least 200 cells/condition. DNA is in blue, centrin in green, and tubulin in red. Scale bar, 10 μm. Error bars, SEM. *p < 0.05; **p < 0.01. Image published in: Cavazza T et al. (2016) © 2016 Cavazza et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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