Cyclin B

Cells were grown on Histogrip (Invitrogen) coated glass coverslips and fixed using ice-cold 100% methanol (β-tubulin) or with 3.7% formaldehyde diluted in PBS with 0.5% Triton X-100 for 10 min (Mad2, pSerCdk, Lamin A/C, Plk1, cyclin B1, and securin). All cells were washed and then blocked (3% BSA, 0,1% Tween 20 in PBS) for 30 min. Cells were incubated with primary antibodies were incubated for 2 h at room temperature in blocking solution. DNA was stained with DAPI. For Lamin A/C staining a Leica DM6000 SP8 confocal with a 63× lens was used. All other images were captured using Leica DM5500 microscope coupled with a Coolsnap HQ2 camera, using a Leica 100× or 40× APO 1.4 lens, powered by Leica LAS AF v3 software. To quantify pSer-CDK, cyclin B and secruin levels in cells, a single in-focus plane was acquired. Using ImageJ (v1.48, NIH), an outline was drawn around each cell and circularity, area, mean fluorescence measured, along with several adjacent background readings. The total corrected cellular fluorescence (TCCF) = integrated density – (area of selected cell × mean fluorescence of background readings), was calculated. This TCCF was then equalized against the mean TCCF of neighboring interphase cells in the same field of view, with results presented as fold increase over interphase levels. Box plots and statistical analysis (2-sided unpaired Student t tests) were performed using GraphPad Prism 5. For all other images, 0.3 µm z-sections were taken, de-convolved, and displayed as 2D maximum projections using ImageJ. False coloring and overlays were performed using Adobe Photoshop CS5 software.

To measure the fluorescent cyclin B1-GFP degradation in living cells, time-lapse images were collected at 1-min intervals. The region was drawn around each cell to be measured, and the identical region was placed in an area without fluorescent objects to be used for background subtraction. The net average fluorescence intensity of a pixel in the region of interest was calculated for each time point. Because cells expressed different levels of fluorescent cyclin B, the net average intensity values were normalized to the initial (first time point) value that was designated as 1. Averages of normalized intensity values of at least five identically treated cells were calculated for each time point and plotted on a graph. For these experiments, all parameters during image acquisition were the same.
To measure fluorescence intensities of MPM2, pS-Cdk, and pNucleolin antibody labeling, 1-μm Z-stacks through cells of different stages of mitosis were acquired. A region was drawn around each cell to be measured, and the same size region was drawn in an area without fluorescent objects to be used for background subtraction. The net integrated intensity for each cell was measured at a single Z plane with highest integrated intensity values in the region of interest (this was usually the plane with the best focus). The weak signal from interphase cells was designated as 1, and the fluorescence intensity values at each mitotic stage were normalized and plotted relative to interphase. Each bar represents an average of 15–30 cells. The intensity of a signal from the control slide labeled with secondary antibodies alone was comparable to the intensity of the background in experimental samples.

For live cell imaging, cells were plated in four-well or eight-well chambered glass-bottom slides (LabTekII) or 24-well glass-bottom plates (MatTek), transfected and imaged in a heated chamber (37°C and 5% CO₂) using a ×10/0.3NA CPlanFLN or ×20/0.5NA UPLFLN objective on a Olympus IX-81 microscope, controlled by Cell-M software (Olympus). Images were acquired using a Hamamatsu ORCA-ER camera and processed using Cell-M software.
For immunofluorescence studies, cells plated on 12-mm coverslips, were pre-extracted with 0.2% Triton X-100 in PEM (100 mM PIPES (pH 6.8), 1 mM MgCl₂ and 5 mM EGTA) for 45 s before fixation with 4% paraformaldehyde in PBS (for Mps1, Mad2 and Hec1). Aurora B and pAurB stainings were performed on cells fixed directly in 4% paraformaldehyde. Coverslips were blocked with 2% bovine serum albumin in PBS/0.1% Triton for 15 min, incubated with primary antibody for 16 h at 4 °C, washed with PBS, and incubated with secondary antibodies and 4,6-diamidino-2-phenylindole for an additional 2 h at room temperature. Coverslips were washed and mounted using ProLong antifade (Molecular Probes). All images were acquired on a DeltaVision RT system (Applied Precision) with a ×100/1.40NA UPlanSApo objective (Olympus) using SoftWorx software. Images are maximum intensity projections of deconvolved stacks.
For quantification of immunostaining, all images of similarly stained experiments were acquired with identical illumination settings and analysed using ImageJ. An ImageJ macro was designed to threshold and select all centromeres and all chromosomes areas (excluding centromeres), using the 4,6-diamidino-2-phenylindole and ACA channels. The convolve filter was first applied to the ACA channel and the threshold selection increased by 1 pixel (to ensure complete kinetochore selection). This was used to calculate the relative mean kinetochore intensity of Mad2, Mps1 and Hec1 ([centromere–chromosome arm intensity (Mad2/Mps1/Hec1)]/[centromere–chromosome arm intensity (ACA)]). For centromere localization of AurB/pAurB, a true background outside the cell was used to subtract from the centromere intensity (and centromere areas were enlarged by 3 pixels to incorporate the more diffuse Aurora B staining).
For quantification of cyclin B–mCherry fluorescence, ImageJ was used to calculate the total integrated fluorescence intensity of individual cells at each time point (after background subtraction of all images using a rolling ball radius of 200 pixels).
For all kinetochore intensity quantifications, means and standard deviations were calculated for each individual experiment and then combined using the standard method for non-overlapping data sets.

Total protein was extracted from A172 cells using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein concentration was quantified with a BCA assay kit (Beyotime Institute of Biotechnology) and equal amount of proteins (20 µg per lane) was separated using 10% SDS-PAGE and subsequently transferred onto PVDF membranes (MilliporeSigma) which were blocked with 5% non-fat milk for 2 h at room temperature. The membranes were incubated with primary antibodies targeting KIF18A (1:2,000; cat. no. ab72417; Abcam), CDK1 (1:10,000; cat. no. ab133327; Abcam), cyclin B (1:50,000; cat. no. ab32053; Abcam), MMP9 (1:1,000; cat. no. ab76003; Abcam), MMP2 (1:1,000; cat. no. ab92536; Abcam), PPP1CA (1:20,000; cat. no. ab52619; Abcam), or GAPDH (1:1,000; cat. no. ab9485; Abcam) overnight at 4˚C. Following the rinse with PBS for three times, membranes were incubated with HRP-conjugated secondary antibodies (cat. no. ab6759; 1:5,000; Abcam) for 1.5 h. The protein bands were visualized using ECL detection reagent (MilliporeSigma) and analysed with ImageJ software 1.8.0 (National Institutes of Health). GAPDH was used as internal reference.