JCS01006 Supplemental Figures

Files in this Data Supplement:

• Figure S1 -

  Fig. S1. Expression of Myc-Mad2 does not appear to compromise the spindle checkpoint. Parental DLD-1 and DLD-1 Myc-Mad2 cells were exposed to nocodazole for 0 hours or 18 hours. The total cell populations were then harvested and analysed by flow cytometry to determine the mitotic index. The bar graph shows that the Myc-Mad2 line accumulates mitotic cells in a similar manner to the parental line.

• Figure S2 -

  Fig. S2. Mad2 localizes to kinetochores after Cenp-E. (A) Immunofluorescence images of DLD-1 Myc-Mad2 cells in different stages of mitosis stained to detect Myc-tagged Mad2 (9E10, red) and Cenp-E (RCE.1, green). The cells were also stained to detect Aurora A (RAA.1, green) in order to determine the spindle orientation. Notice that, because the antibodies were used to detect Cenp-E and Aurora A are from the same species, both proteins appear in the same channel. However, the spindle poles are easily distinguished from kinetochores owing to their larger size. (B) Asynchronous DLD-1 Myc-Mad2 cells were fixed and stained to detect DNA, centromeres/kinetochores (ACA, blue), Cenp-E (green) and Myc-Mad2 (red). The cells were then analysed by deconvolution microscopy and image stacks projected onto a single plane. The images show that Cenp-E is detectable at kinetochores prior to Myc-Mad2. Scale bar, 5 mm. Quantitation of pixel intensities is shown in Fig. 1E.

• Figure S3 -

  Fig. S3. Repression of Bub1 does not prevent accumulation of mitotic cells following exposure to Taxol. DLD-1 cells were transfected with control and Bub1 siRNA duplexes. 48 hours after transfection the cells were incubated with Taxol, and the number of mitotic cells scored by fluorescence microscopy after 1 hour, 3 hours and 6 hours. At least 1000 cells were counted for each time point and values represent the mean and s.e.m. from at least three independent experiments.