Fig. 2

PIP4KIIγ is required for assembly of bipolar spindles. a, b Depletion of PIP4KIIγ induces defects in spindle assembly. CGL2 cells depleted of PIP4KIIα, β, or γ using specific shRNA (a) or siRNA (b) were then fixed and stained for mitotic spindles with antibodies against α-tubulin and γ-tubulin. The percentage (mean ± SD) of mitotic cells containing abnormal mitotic spindles was determined using at least 500 mitotic cells from three independent experiments. **p < 0.01 by Student’s t test compared to the control depleted cells. c Representative images of the mitotic spindle immunofluorescence-stained for α-tubulin (red), γ-tubulin (green), and chromosome (blue) in PIP4KIIγ-depleted culture. i, the normal bipolar spindle. ii and iii, the bipolar spindle with mis-aligned chromosomes. iv, the multipolar spindle. d The depletion efficiency of PIP4KIIγ by specific shRNA. e Representative images of the mitotic spindle after nocodazole treatment and washout. The control (pLKO) or cells depleted of PIP4KIIγ (sh2C) were treated with 3 μM Nocodazole for 3 h. Nocodazole was then completely washed out, and the cells were incubated in drug-free medium for the indicated time before being immediately fixed and immunostained for α-tubulin (red) and γ-tubulin (green). The chromosomes were counterstained with DAPI (blue). f The number of MT asters in cells. The results are the distribution and mean ± SD of the aster numbers in at least 250 mitotic cells for each condition, as determined from three experiments