Figure 7.

Pericentrin is crucial for bipolar spindle elongation in cells with two centrosomes. (A) Mitotic spindle structures upon treatment with siRNA in cells with two centrosomes. Red and blue represent α-tubulin and DNA, respectively. Z-projections: 21 planes, 1 µm apart. Scale bar, 5 µm. (B) Quantification spindle length of HeLa cells (n = 6, triplicates, two independent experiments, at least 15 spindles in each assay). Line and error bars represent the mean and SD. Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.01. (C) CEP192 observed in two-centrosome cells. Green, red, and blue represent GT335, CEP192, and DNA, respectively. Z-projections: 20 planes, 0.5 µm apart. Scale bar, 10 µm. (D) The signal intensity of CEP192 on centrosomes in C. Line and error bars represent the mean and SD (n ≥ 50 cells from two independent experiments). Kruskal–Wallis test was used to determine the significance of the difference. *, P < 0.0001; n.s., not significantly different. (E) Time-lapse observation of the structure of microtubules upon depletion of pericentrin in two-centrosome HeLa cells were observed with a 40× objective. Gray represents SiR-tubulin. Z-projections: 10 planes, 2.2 µm apart. Scale bar, 10 µm. Time zero corresponds to mitotic onset. (F) Averaged time courses of the pole length at each time point in E. The length between two poles of spindle was measured from 40 cells from two independent experiments. Time course data were aligned at the time of the mitotic onset (0 min). Error bars represent SD. (G) Mitotic spindle structures of two-centrosome A549 and U2OS cells. Red and blue represent α-tubulin and DNA, respectively. Z-projections: 31 planes, 0.5 µm apart. Scale bar, 10 µm. The spindle length of A549 (H) and U2OS (I) cells upon depletion of pericentrin (n > 40 from two independent experiments). Line and error bars represent the mean and SD. The Mann–Whitney U test (two tailed) was used to obtain a P value. *, P < 0.005; **, P < 0.0001.