Supplmentary Materials

This article contains the following supporting material:

• Supplementary Figure 1 - Exogenous FL^1-433 protein levels can be titrated by the dilution of adenovirus. (A) BS-C-1 cells were infected with FL^1-433 adenoviral dilutions of 1:100, 1:500, 1:1000, 1:2000, 1:3000, 1:4000 or 1:5000 and harvested for immunoblots at 18 hours post infection. The cyclin B1 western shows that at the 1:5000 dilution, FL^1-433 levels are comparable to endogenous cyclin B1. (B) At the 1:500 dilution, chromatin, centrosome, spindle microtubule and kinetochore signal are present with a maximum intensity value of 3144. (C) At the 1:5000 dilution, chromatin, centrosome, spindle microtubule and kinetochore localization can still be observed, with a maximum intensity of only 1315. The minimum values in both images are comparable (214 for 1:500, 234 for 1:5000). This reduced maximum intensity value in the 1:5000 dilution contributes to a somewhat grainy appearance of the image as compared to the image in (B).
• Supplementary Figure 2 - Pre-absorption of the GNS1 antibody with recombinant cyclin B1-CDK1 ablates endogenous kinetochore signal. BS-C-1 cells were processed for immunofluorescence with control (A-C) and pre-absorbed (D-F) antibodies. The GNS1 antibody used in Figure 2 and 4 was pre-absorbed against recombinant cyclin B1-CDK1. ACA and control antibody signal was not diminished whereas pre-absorption significantly decreased cyclin B1 signal at kinetochores (D). A scale bar representing 10 µm is shown in (A).
• Supplementary Figure 3 - Positive cyclin B1 signal is not bleed through from the Hec1 channel. (A) An anaphase cell, as indicated by its DNA morphology, is positive for ACA (B) and Hec1 (C) but not cyclin B1 (D). Images from an anaphase cell demonstrate that cyclin B1 signal is absent even when Hec1 is present, demonstrating that cyclin B1 signal originates from the cyclin B1 protein rather than Hec1 signal.
• Supplementary Figure 4 - Hec1 signal correlates with cyclin B1 levels in siRNA transfected cells. Positive signal demonstrates specificity for the cyclin B1 antibody and immunofluorescence protocol. (A-F) In cells transfected with siRNA against Hec1, we observed variation in the level of Hec1 depletion (as shown in A). In this scenario, cyclin B1 signal (E) correlates with Hec1 signal (D), demonstrating that the absence of cyclin B1 signal at kinetochores following Hec1 depletion (right cell, E) is not due to a failed immunofluorescence procedure.
• Supplementary Video 1 - This video shows a ^FL1-433 infected BS-C-1 cell from prophase through degradation of ^FL1-433 at the metaphase to anaphase transition. Images were collected every 30 seconds. Images in Figure 1 are taken from this movie.
• Supplementary Video 2 - This video shows a FL^1-433 infected BS-C-1 treated with 100 µg/mL monastrol. Note that the centrosomes coalesce and chromosomes are not organized along a central metaphase plate. Images were collected every 30 seconds.
• Supplementary Video 3 - This video shows a CT^108-433 infected BS-C-1 cell from prophase through metaphase completion. Images were collected every 30 seconds. Note that Figure 7 shows that FL^1-433 and CT^108-433 signal at kinetochores is equivalent.
• Supplementary Video 4 - This video shows a FL^Y170A infected BS-C-1 cell from prophase through late prometaphase. Images were collected every 45 seconds. Kinetochore localization becomes apparent at 1:30, but is clearest at 4:30 and is sustained throughout the duration of the movie. Note that Figure 7 shows that FL^1-433 and FL^Y170A signal at kinetochores is equivalent.