Figure 4: Impact of gatastatin on mitotic spindle dynamics.

(a,b) γ-Tubulin function is important for normal bipolar spindle formation. (a) Scheme of the experiment in b. HeLa cells were arrested at prometaphase by treatment with 10 μM of STLC for 15 h before STLC was washed out. Progression through mitotic phases was then monitored either in the presence or absence of 30 μM of gatastatin for 2 h. (b) The mitotic stages of 20 cells per experiment from the scheme in a were analysed in three independent experiments. Error bars represent s.d. (c–e) γ-Tubulin function is important for normal bipolar spindle maintenance. (c) Scheme of the experiment in d,e. After STLC washout, HeLa cells were arrested at metaphase with bipolar spindles by treatment with 5 μM of MG132 for 2 h. 30 μM Gatastatin was added to cells and incubated for 2 h in the presence of MG132. (d) The spindle length (distance between centrosomes) of cells in scheme c was measured as described in Fig. 3g. Spindles of 20 cells per experiment were analysed. Three independent experiments were performed and one representative experiment is shown. Error bars represent s.d. Two-tailed, unpaired Student’s t-test was used to obtain P value. (e) The effect of gatastatin on spindle MT dynamics was analysed using HeLa cells expressing EB3-EGFP. EB3 signals of spindle in c were monitored. EB3 tracks are displayed in rainbow colours as maximum intensity projection of all time points from a 60-s sequence of imaging. Scale bar, 5 μm. MT parameters are summarized in Table 4.