Volume 22, Issue 6, Pages 1424-1438 (February 2018) BUB1 Is Essential for the Viability of Human Cells in which the Spindle Assembly Checkpoint Is Compromised  Jonne A. Raaijmakers, Roy G.H.P. van Heesbeen, Vincent A. Blomen, Louise M.E. Janssen, Ferdy van Diemen, Thijn R. Brummelkamp, René H. Medema  Cell Reports  Volume 22, Issue 6, Pages 1424-1438 (February 2018) DOI: 10.1016/j.celrep.2018.01.034 Copyright © 2018 The Author(s) Terms and Conditions

Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions

Figure 1 HAP1 Cells Do Not Require a Functional SAC (A) Protein lysates from WT HAP1 cells, ΔMAD1 clone 1 and 2, and ΔMAD2 clone 1 and 2 were analyzed by western blot. SMC1 was used as loading control. Asterisk marks crossreacting band. Only data from ΔMAD1 c2 and ΔMAD2 c1 are shown for the more detailed characterization in the rest of the figure. (B) Immunofluorescent (IF) images of WT, ΔMAD1, and ΔMAD2 cells treated with nocodazole and MG132 for 3 hr before fixation. Cells were stained for either MAD1 or MAD2 (depicted in green). Centromeres/anti-centromere antibodies (ACAs) are depicted in red and DNA in blue. Scale bar, 10 μM. (C) Average time in mitosis as determined by time spent from NEB until anaphase for WT (n = 60), ΔMAD1 c2 (n = 60), and ΔMAD2 c1 cells (n = 43) from 2 independent experiments. Error bars represent 95% confidence intervals. (D) Average time in mitosis of cells treated with 250 ng/mL nocodazole as determined by time from NEB until chromosome decondensation or death in mitosis as determined by DNA hypercondensation and fragmentation for WT (n = 32), ΔMAD1 c2 (n = 30), and ΔMAD2 c1 cells (n = 25). Error bars represent 95% confidence intervals. (E) Amount of chromosome missegregations in WT (n = 104), ΔMAD1 c2 (n = 54), and ΔMAD2 c1 (n = 114) cells. Missegregations were determined on fixed cells stained with DAPI and pHistone H3-Ser10. (F) Growth assay of WT, ΔMAD1 c2, and ΔMAD2 c1 cells treated for 7 days with increasing doses of Taxol or nocodazole. Cells were fixed with methanol and stained with crystal violet. See also Figure S2. Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions

Figure 2 Haploid Genetic Screen Identifies Factors Involved in Chromosome Congression (A) Schematic representation of insertional mutagenesis using a gene trap virus. (B–D) Representative plots of three individual screens for WT (replicate 3) (B), ΔMAD1 clone 1, and ΔMAD2 clone 2 (C), and ΔMAD2 clone 2 (D). Each dot represents an individual gene. Number of integrations is plotted on the x axis. Percentage of sense integrations is plotted on the y axis. Highlighted genes are scored as significantly synthetic lethal with loss of the SAC. (E) Functional interaction network for hits as determined by search tool for recurring instances of neighboring genes (STRING) analysis revealed a strong enrichment for kinetochore-associated proteins and for the condensin II complex. The thickness of the line represents the strength of the evidence for the specific interactions. See also Figure S3. Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions

Figure 3 BUB1 Regulates Chromosome Congression and Only Has a Minor Role in the SAC (A) Average percentage of sense integrations mapped to the BUB1 and BUBR1 genes from all screens in WT, ΔMAD1, and ΔMAD2 cells. Error bars represent SD. (B) WT and ΔBUB1 cells were analyzed by western blot. α-Tubulin was used as a loading control. (C) IF images of WT and ΔBUB1 cells treated with nocodazole for 3 hr before fixation. Cells were stained for BUB1 (green), centromeres/ACA (red), and DNA (blue). Scale bar, 10 μM. (D) IF images of WT and ΔBUB1 cells treated with nocodazole for 3 hr before fixation. Cells were stained for BUB1 (green), BUBR1 (red), and centromeres/ACA (blue). Scale bar, 10 μM. (E) Growth assay of WT and ΔBUB1 cell, treated for 7 days with increasing doses of the MPS1 inhibitor Cpd-5. Cells were fixed with methanol and stained with crystal violet. (F) Time from NEB till metaphase and from metaphase till anaphase was determined for WT (n = 46) and ΔBUB1 (n = 50) cells in 2 independent experiments. DNA was visualized using SiR-DNA. Error bars indicate 95% confidence intervals. (G) WT and ΔBUB1 cells expressing H2B-RFP were transfected with siGAPDH (−) (n = 84 and 61 cells) or siMAD2 (+) (n = 83 and 58 cells) for 24 hr before filming. Total time from NEB to anaphase was scored. Error bars indicate 95% confidence intervals. (H) Quantification of the percentage of cells from (G) displaying chromosome missegregations in anaphase. Error bars indicate SD. (I) Average time in mitosis of cells treated with nocodazole. DNA was visualized by SiR-DNA. Bars represents time from NEB until chromosome decondensation or death in mitosis for WT (n = 43) or ΔBUBR (n = 56) cells from 3 independent experiments. Error bars represent SD. Student’s t test was performed for statistical testing. (J) Average time in mitosis of cells treated with nocodazole + 200 nM reversine. n > 50 cells/condition from 3 independent experiments. Error bars represent SD. (K) A schematic depiction of the different BUB1 constructs used in this experiment. WT and ΔBUB1 cells were either untransfected or transfected with the indicated constructs 48 hr before start of the movie. 200 nM reversine was added at the start of the movie. DNA was visualized using SiR-DNA, and time in mitosis was determined from 2 independent experiments. Error bars indicate SEM. See also Figure S4. Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions

Figure 4 Mislocalization of Aurora B Does Explain Chromosome Alignment Defect in ΔBUB1 Cells (A) Protein lysates from WT and ΔBUB1 cells were analyzed for pH2A-T120 levels by western blot. α-Tubulin was used as a loading control. (B) IF images of WT and ΔBUB1 cells treated with nocodazole for 3 hr before fixation. Cells were stained for pH2A-Thr120 (green), centromeres/ACA (red), and DNA (blue). Scale bar, 10 μM. (C) Cells were treated as in (B) but stained for total Aurora B (green), centromeres/ACA (red), and DNA (blue). (D) IF images of WT and ΔBUB1 cells either untransfected or transfected with the indicated constructs 48 hr before fixation. Cells were treated as in (B) and stained for pH2A (red) and DNA/DAPI (blue). Scale bar, 10 μM. The average level of pH2A relative to the average DAPI signal was determined from maximum projections for < 10 cells per condition in 2 independent experiments. Transfected cells were selected based on their GFP-positivity. Error bars represent SD. (E) Cells were treated as in (D) but stained for Aurora B (red) and DNA (blue). Scale bar, 10 μM. Graph depicts average Aurora B levels as determined by measuring Aurora B intensity by performing at least 15 individual line scans of 15 μm spanning a centromere. Transfected cells were selected based on their GFP positivity. (F) Time from NEB until metaphase was determined for WT tetraploid HAP1 cells or ΔBUB1 tetraploid HAP1 cells either untransfected or transfected with the indicated constructs 48 hr before the start of the movie. A schematic overview of the two different BUB1 constructs (WT and D946N) used in this experiment is depicted next to the graph. DNA was visualized using SiR-DNA. For every condition, >64 cells were quantified in 3 independent experiments. Error bars indicate SEM. Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions

Figure 5 BUB1 Does Not Regulate Chromosome Congression through the RZZ Complex (A) Average percentage of sense integrations in the genes of the RZZ complex and its upstream factor, KNL-1, in the WT, ΔMAD1, and ΔMAD2 screens. (B) IF images of WT and ΔROD cells treated with nocodazole for 3 hr before fixation. Cells were stained for ZW10 (green), centromeres/ACA (red), and DNA (blue). Scale bar, 10 μM. (C) Cells were treated as in (B) but stained for Spindly (green) instead. Scale bar, 5 μM. (D) WT and ΔROD cell lysates were analyzed for ROD protein levels. Asterisks mark crossreacting bands. (E) Growth assay of WT and ΔROD cells treated for 7 days with increasing doses of the MPS1 inhibitor Cpd-5. Cells were fixed with methanol and stained with crystal violet. (F) Quantification of time in mitosis for WT (n = 60) and ΔROD (n = 60) cells expressing H2B-RFP, from 2 independent experiments. Time from NEB to alignment and alignment to anaphase was determined. Error bars indicate SD. (G) WT and ΔROD cells expressing H2B-RFP were transfected with siGAPDH (−) or siMAD2 (+) for 24 hr before filming. Total time from NEB to anaphase was scored. n = 60 cells/condition from 2 independent experiments. Error bars indicate SD. (H) Quantification of the amount of missegregations from the cells in (G). Error bars indicate SD. (I) Average time in mitosis of WT (n = 87) and ΔROD (n = 85) cells expressing H2B-RFP treated with nocodazole. Time was determined by calculating time from NEB until chromosome decondensation or death in mitosis. Two independent experiments were performed. Error bars represent SD. (J) IF images of WT and ΔROD cells treated with nocodazole and MG132 for 3 hr before fixation. Cells were stained for MAD2 (green), centromeres/ACA (red), and DNA (blue). Scale bar, 5 μM. (K) IF images of WT and ΔBUB1 cells treated with nocodazole for 3 hr before fixation. Cells were stained for ZW10 (green), centromeres/ACA (red), and DNA/DAPI (blue). Scale bar, 5 μM. (L) Time from NEB until metaphase was determined for WT tetraploid HAP1 cells or ΔBUB1 tetraploid HAP1 cells either untransfected or transfected with the indicated constructs 48 hr before the start of the movie. A schematic overview of the two different BUB1 constructs (WT and Δ437–521) used in this experiment is next to the graph. DNA was visualized using SiR-DNA. For every condition, >64 cells were quantified in three independent experiments. Error bars indicate SEM. See also Figure S5. Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions

Figure 6 The BUB1 C-Terminal Tail Regulates Chromosome Congression and Recruits CENPF (A) Time from NEB until metaphase was determined for WT tetraploid HAP1 cells or ΔBUB1 tetraploid HAP1 cells either untransfected or transfected with the indicated constructs 48 hr before the start of the movie. DNA was visualized using SiR-DNA. Data are from 3 independent experiments, and >40 cells were analyzed per condition. Error bars indicate SEM, except for the BUB1 1–778 construct, where 26 cells were analyzed from a single experiment and error bars indicate 95% confidence intervals. (B) IF images of WT and ΔBUB1 cells either untransfected or transfected with the indicated constructs 48 hr before fixation. Cells were treated with nocodazole 3 hr before fixation and stained for CENPF (red) and centromeres/ACA (blue). Scale bar, 10 μM. Relative GFP and CENPF levels at kinetochores were determined from maximum projections for all kinetochore pairs in a cell using an automated macro in ImageJ for >15 cells/condition from two independent experiments. Background correction was performed by subtracting cytoplasmic levels. Error bars represent SD. (C) WT and ΔCENPF cells were harvested, and CENPF protein levels were analyzed by western blot. Ponceau S staining was used as a loading control. (D) Time from NEB until metaphase was determined for WT or ΔCENPF cells. DNA was visualized using SiR-DNA. For every condition, >100 cells were quantified from three independent experiments. Error bars indicate SEM. See also Figure S6. Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions

Figure 7 BUBR1 Is Essential for the SAC but Does Not Regulate Chromosome Alignment in HAP1 Cells (A) WT and ΔBUBR1 cells were harvested, and BUBR1 protein levels were analyzed by western blot. Ponceau S staining was used as a loading control. (B) IF images of WT and ΔBUBR1 cells treated with nocodazole for 3 hr before fixation. Cells were stained for BUBR1 (green), centromeres/ACA (red), and DNA/DAPI (blue). Scale bar, 10 μM. (C) Average time in mitosis of cells treated with nocodazole. DNA was visualized by SiR-DNA. Time was determined by calculating time from NEB until chromosome decondensation or death in mitosis for WT (n = 52) or ΔBUBR1 (n = 47) cells from 2 independent experiments. Error bars represent SD. (D) Representative images of WT and ΔBUBR1 cells treated with MG132 for 1 hr prior to fixation. Cells were stained with tubulin (red), DAPI (blue), and pH3(Ser10) (green). Right: percentage of mitotic cells displaying full chromosome alignment in fixed WT (n = 109 cells) and ΔBUBR1 (n = 104 cells) treated with MG132 for 1 hr to prevent mitotic exit. (E) Time from NEB till metaphase was determined for WT (n = 17), ΔBUB1 (n = 24), and ΔBUBR1 (n = 20) cells. 20 μM proTAME was added at the start of the movie to prevent premature mitotic exit. DNA was visualized by SiR-DNA. Significance was determined by a Student’s t test. (F) WT, ΔBUB1, and ΔBUBR1 cells were transfected with non-targetable siRNA (−) or MAD2 siRNA (+) 24 hr before start of the movie. DNA was visualized using SiR-DNA, and time from NEB to alignment and alignment to anaphase was determined. For each condition >50 cells were scored in two independent experiments. (G) Quantification of the percentage of cells from (F) displaying chromosome missegregations in anaphase. Error bars represent SD. See also Figure S7. Cell Reports 2018 22, 1424-1438DOI: (10.1016/j.celrep.2018.01.034) Copyright © 2018 The Author(s) Terms and Conditions