Fig 2: Analysis of In Vitro Ubiquitylation Activities of APC/C Lacking APC16 and/or APC7(A) Ubiquitylation activity of purified APC/C variants toward securin. APC/C was purified from APC8-mCherry (WT), ΔAPC7 APC8-mCherry (ΔAPC7), or ΔAPC16 APC8-mCherry (ΔAPC16) cells using an antibody against APC3 and incubated for different times with recombinant securin supplemented with UBE2C and UBE2S as indicated. Ubiquitylation of securin was analyzed by an α-securin immunoblot. A representative result from two experiments is shown. WT, wild-type; DN, dominant-negative.(B) Immunoblot analysis of the purified APC/C used for Figure 2A. Input, supernatant, and immunoprecipitated fractions (immunoprecipitation [IP]: APC3) from the indicated cell lines were analyzed with the indicated antibodies.(C) Ubiquitylation activity of purified APC/C variants toward cyclin B1 as described in (A). Ubiquitylation of cyclin B1 was analyzed by an α-cyclin B1 immunoblot. A representative result from two experiments is shown.(D) Immunoblot analysis of the purification of APC/C used for Figure 2C, analyzed as described in (B).See also Figure S1.

Fig 3: Loss of APC7 or APC16 Provides Synthetic Viability to MAD2 Deletion(A) Strategy for assessing the synthetic viability of MAD2 deletion with the ΔAPC7 genotype. Equal numbers of wild-type, APC8-mCherry, and APC8-mCherry ΔAPC7 cells were mixed and seeded together, resulting in a mixed cell population with one-third of cells lacking APC7. The genomic MAD2 locus was then targeted by CRISPR/Cas9 using a drug-selectable donor plasmid designed to disrupt the MAD2 gene. After selection, each clonal cell line was analyzed by immunoblotting to assess loss of MAD2 and to determine its parent-of-origin cell line.(B) Analysis of APC7 and MAD2 expression in the six clonal ΔMAD2 cell lines retrieved from the setup outlined in (A). GAPDH levels served as a loading control. Note that all six retrieved clonal cell lines derive from APC8-mCherry ΔAPC7 cells.(C) Scheme of the CRISPR/Cas9-based MAD2-GFP synthetic viability assay. Cells expressing endogenously GFP-tagged MAD2 (MAD2-GFP) were transfected with a drug-selectable deletion cassette for the MAD2 gene along with a drug selection-less plasmid encoding a guide RNA targeting either APC7 or APC16. After drug selection, cell colonies were microscopically inspected for loss of MAD2-GFP fluorescence. Cell colonies lacking green fluorescence were then analyzed by immunoblotting for loss of MAD2 expression. The status of APC7 and APC16 in MAD2-deficient cells was assessed by immunoblotting or sequencing of the genomic locus, respectively.(D) Immunoblot analysis of the four clones retrieved from the MAD2-GFP synthetic viability assay performed in combination with an APC7 targeting guide RNA. Note that all retrieved clonal cell lines lost expression of APC7.(E) Analysis of SAC functionality in ΔAPC7ΔMAD2 and ΔMAD2-GFPΔAPC16 cells. Cellular DNA from wild-type, ΔAPC7ΔMAD2, and ΔMAD2-GFPΔAPC16 cells, with or without 18 hr of 200 nM nocodazole, was stained with propidium iodide and analyzed by flow cytometry. MAD2 depletion by RNAi was performed in wild-type HCT116 cells for comparison. The tables at the top show the percentage of cells with the respective (2N, 2N-4N, 4N, and >4N) DNA content. The bottom panel shows the corresponding flow cytometry profiles from the indicated cell lines.See also Figure S4.

Fig 4: Cdc20 phosphorylation prevents interphase APC/C binding.(a,b) Stable HeLa cell lines expressing the indicated YFP-tagged Cdc20 proteins were depleted of endogenous Cdc20 and 8 h after release from a thymidine block YFP–Cdc20 complexes were purified. The samples were analysed by SDS–PAGE and probed for APC1, APC3 and APC7 and this was quantified by LI-COR technology. (c) Bar diagram shows the median and range from two (for APC1 and 7) or three (for APC3) independent experiments. Signals were normalized to Cdc20 and Cdc20 WT. (d,e) As in a,b but YFP-tagged Cdc20 WT cells were treated after 7.5 h for 30 min with Calyculin A and then collected. Representative of two independent experiments. (f) Stable HeLa cell lines expressing the indicated YFP-tagged Cdc20 proteins were synchronized as outlined in Fig. 3a and following release from the last thymidine block followed by time-lapse microscopy. The bar graph shows cells entering mitosis after normalization to Cdc20 WT-expressing cells. Average of two independent experiments (at least 127 cells for each condition) with range indicated by the lines. (g) Stable HeLa cell lines expressing YFP–Cdc20WT or YFP–Cdc20Ala were synchronized as outlined in Fig. 3a and after release from the last thymidine block mitotic entry was monitored by time-lapse microscopy. In these experiments, Cdc20 was not depleted but cells instead treated with a control siRNA (luciferase (ctrl)) or a siRNA-targeting APC3. Average of three experiments (at least 312 cells in each condition) is indicated in the bar graph with s.d. indicated by the line. An unpaired t-test without assuming equal s.d. was used for statistical comparison (***P<0.0001; NS, non-significant).

Fig 5: Kinetochore localization of the Bub1-independent pool of BubR1/Bub3 stimulates checkpoint signalling.(a) Cartoon of the internal binding between Bub3 and GLEBS domain on BubR1 in the fusion protein BubR1–Bub3. Venus-tagged RNAi-resistant wild-type (WT) BubR1 or BubR1–Bub3 plasmid was transfected into HeLa cells together with RNAi oligos against BubR1 for 48 h. After nocodazole treatment, mitotic cells were collected and lysed. Immunoprecipitation was performed against Venus tag by GFP-Trap beads. The presence of APC7, Bub3, Cdc20 and Mad2 was analysed by Li-cor quantitative western blot. (b) Western blot results in a were quantified and plotted. The values from BubR1 WT were set to 1. (c) Venus-tagged BubR1 WT or BubR1–Bub3 or BubR1 GLEBSmut–Bub3 plasmid was transfected into HeLa cells for 48 h. After nocodazole treatment, mitotic cells were collected and lysed and extract separated on a Superose-6 column and the relevant fractions analysed by western blot. Migration of molecular weight standards (670 kDa) or calculated molecular weights based on a calibration curve is indicated on top. (d) HeLa cells were co-transfected with BubR1 RNAi oligos and Venus-tagged BubR1–Bub3, BubR1–Bub3 2E, BubR1Δ440–460–Bub3 or BubR1Δ440–460–Bub3 2E plasmid. Live-cell imaging was performed to record the time cells spent in mitosis in the presence of low-dose nocodazole. Representative still images are shown. Scale bar, 5 μm. (e) Quantification of the live-cell imaging results of d. (f) HeLa cells were co-transfected with BubR1 RNAi oligos and Venus-tagged BubR1Δ440–460–Bub3 or BubR1Δ440–460–Bub3 2E plasmid. Live-cell imaging was performed to record the time cells spent in mitosis in the presence of taxol. Quantification of the live-cell imaging results is shown. n indicates the total number of cells counted. The red line in the plot indicates median time cells spent in mitosis. A Mann–Whitney test was used for statistical comparison of the different samples. (****P≤0.0001). Note: the red dots in f represent the cells still arrested in mitosis when filming stopped.