Fig 2: Premature centriole separation in CDC20-depleted cells.(A) Immunoblot analysis was performed to determine the CDC20 levels in siCDC20-transfected RPE-1 cells. (B) The CDC20-depleted mitotic cells were immunostained with antibodies specific to centrin-2 (green) and CEP135 (red). Scale bar, 10 μm. (C) The CDC20-depleted RPE-1 cells were synchronized with a thymidine block and release, and fixed for immunostaining at the indicated time points. The control cells were treated with STLC for M phase arrest. The number of mitotic cells with separated centrioles was counted at each time points. Experiments were repeated three times with over 150 cells per each group. Values are means and standard deviations. (D) HeLa cells were treated with the STLC (10 μM) for 20 h and their chromosomes were spread and stained with DAPI.

Fig 3: The level of the complex formed between CDC20 and MAD2 in response to nocodazole or taxol treatment at various stages of the cell cycle.(a) HeLa cells were treated with 200 nM nocodazole (Noc) or 30 nM taxol for 12 hours and fixed with cold methanol before they were examined using PLA for CDC20 and MAD2 interaction (in red). DNA was stained with DAPI (grey in the top images and blue in the merged images). Scale bar = 50 μM. White dash lines showing #1 & 2 cells: example of nocodazole arrested prometaphase-like cells. (b) Comparison of the quantitative results of the average fluorescent intensities of CDC20-MAD2 from individual cells. ProM: Normal prometaphase, Noc: Nocodazole. Cell number used for quantifications: ProM (n = 40), 200 nM Noc ProM (n = 120), 30 nM taxol ProM (n = 139). (c) Quantitative results of the average fluorescent intensities of CDC20-MAD2 interaction from individual cells after treated with 200 nM nocodazole at time intervals as indicated. *P < 0.015, ****P < 0.0001. n: The number of cells used for quantification.

Fig 4: The interaction between CDC20 and MAD2 is cell cycle regulated with the levels high during mitosis, but it is also detectable during anaphase in unperturbed HeLa cells.Projected Z-stack sectional confocal images showing examples of the PLA signals between CDC20 and MAD2 (a), but not between pairs of MAD2-Random IgG (b) and CDC20-Random IgG (c) at selected cell cycle stages. White and yellow dash circle lines highlight the cytoplasmic region and the nuclear region of a prophase cell respectively. Arrows and arrowheads highlight the fluorescent signals detected in the nucleus and cytoplasm respectively. DNA (in blue) and microtubule (in green) morphologies were used to determine the cell cycle stages. (d) HeLa cells were stained with pericentrin antibody to mark the centrosomes (in green). DNA (blue) and centrosome morphologies were used to determine the cell cycle stages and this allows the separation of the cell cycle stages before prophase into interphase, late G2 and early prophase as indicated. The white dash circle lines highlight the cell boundaries and the arrows highlight the centrosomes. (e) Showing the quantitative profile of the interaction between CDC20 and MAD2 illustrated by the average PLA fluorescence intensities from entire cellular regions (white encircled dash lines indicated in a & b) of individual cells at different cell cycle stages. n: The number of cells used for quantification. ****P < 0.0001. Standard deviation bars are in red. The images were produced using a Leica TCS SP2 confocal system using sequential + Z-stack scanning mode with consistent system settings. Int: Interphase, L-G2: Late G2, Pro: Prophase, ProM: Prometaphase, Met: Metaphase, Ana: Anaphase, Telo: Telophase. Scale bar = 5 μM.

Fig 5: CDC20-MAD2 complex fluorescent signal levels correlate with the levels of cyclin B1 in HeLa cells treated with 200 nM nocodazole.(a) Examples of projected confocal images produced from relevant Z-stacks revealing three different cells with correlation between different levels of cyclin B1 (green) and CDC20-MAD2 PLA signals (red). Cyclin B1 protein levels (green) were detected using a rabbit anti-cyclin B1 (Santa Cruz, H-433, sc-752) staining after the completion of the PLA of CDC20-MAD2 (in red). The average fluorescent intensities (units in a.u.) of CDC20-MAD2 from the regions with dashed lines encircling these three cells were shown above the images. The higher level of cyclin B1 correlates with higher numbers of the CDC20-MAD2 complex. DNA was stained with DAPI (in grey or blue in merged images). Scale bar = 5 μM. (b) Diagrams showing the correlations between the average fluorescent intensities of cyclin B1 and the average fluorescent PLA signals of CDC20-MAD2 quantified from individual arrested cells after 12 hours treatment with 200 nM nocodazole as described in A. XY frequency distribution from PRISM was used to assess the significance of the correlations between two groups as indicated. The P value was < 0.0001, showing strong correlation. n: The number of cells used for quantification.