DEV035089 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Concentration dependence of monastrol-induced spindle collapse in mouse eggs. Left graph (A) shows plots of average spindle length at times indicated following addition of monastrol at the concentration indicated (arrow). Each curve is a composite of at least five spindles. Right graph (A′) shows concentration dependence of the initial velocity of spindle collapse. The initial velocity of spindle collapse was calculated as described in the Materials and methods. Error bars are s.e.m.

• Supplemental Figure S2 -

  Fig. S2. Timelapse imaging of monastrol-induced spindle collapse in mitotic one-cell embryos. One-cell embryos were allowed to undergo nuclear envelope breakdown, and then transferred to MG132 for 1-2 hours before imaging, to allow the mitotic spindle to be built. Imaging was performed using MAP7-GFP (green), Hoechst (blue), and epifluorescence microscopy, similar to the Egg experiment presented in Fig. 2C. 200 µM monsatrol was added at t=0.

• Supplemental Figure S3 -

  Fig. S3. Monastrol prevents spindle formation in blastocysts. (A) Blastocysts were treated with 10 µM nocodazole for 2 hours, then transferred to media containing either 25 µM MG132 (control group, left), or MG132 plus 200µM monastrol (right). Embryos were fixed and immunostained for microtubules (red) and chromosomes labelled with Hoechst (blue), and images were taken using epifluorescence. Note the presence of healthy bipolar spindles in controls (left, nocodazole, followed by MG132 only), and the presence of collapsed spindles in the monastrol-treated group (right). (B) Quantification of this experiment. Note that monastrol impedes correct spindle assembly in almost all cells, indicating that the drug could access the mitotic machinery in blastocysts, and confirming the role of kinesin 5 in spindle assembly.

• Supplemental Figure S4 -

  Fig. S4. Increased monastrol concentration does not increase the proportion of metaphase spindles that collapse in blastocysts. Metaphase spindles were exposed to 200 or 400 µM monastrol for 30 minutes. Quantification of the proportion of spindles that collapsed is shown. Doubling the concentration of monastrol had no effect upon the proportion of spindles that remained bipolar (P>0.85).