Fig. 3

Spindle pole-associated MTs are less stable in PIP4KIIγ-depleted mitotic cells. a A representative image frame of a time-lapse sequence from a cell stably expressing EGFP-EB1 (HeLa-EB1-GFP). b Depletion of PIP4KIIγ reduces the nucleation of EGFP-EB1 comets. The control depleted (pLKO) or PIP4KIIγ-depleted (sh2C) HeLa-EGFP-EB1 cells were subjected to time-lapse imaging under a confocal microscope as described in “Methods” section. Typically, each comet was visible for three to five frames. Results are the mean ± SD of EB1 comets nucleated from at least 100 spindle poles in three independent experiments. *p < 0.05 by Student’s t test compared to the control (pLKO). c, d Depletion of PIP4KIIγ shortens cold-resistant K-MT. Representative images of the mitotic spindle after cold treatment (c). The control (pLKO) or PIP4KIIγ-depleted (sh2C) CGL2 cells were subjected to cold treatment for 30 min before being stained for mitotic spindles as described in Fig. 2c. Arrowheads indicate the spindle poles. The percentages of mitotic cells with long (blanked bar) or short (dotted bar) cold-resistant K-MTs (d). e, f Depletion of PIP4KIIγ disorganizes the spindle pole-associated cold-resistant K-MT. Representative images of the mitotic spindle after monastrol and cold treatment (e). The control (pLKO) or PIP4KIIγ-depleted (sh2C) CGL2 cells were first treated with 50 μM monastrol for 4 h to prevent centrosome separation and then subjected to cold treatment for 30 min before being stained for mitotic spindles as described in Fig. 2b. The percentages of mitotic cells with well-organized (blanked bar) or disorganized (dotted bar) spindle pole-associated cold-resistant K-MT (f). Results are the mean ± SD of at least 250 mitotic cells from three independent experiments. **p < 0.01 by Student’s t test compared that in the control. Scale bar, 10 μm