DLD1/Reference a" 2N 2N 2N/DLD1 2N/ /DLD1 c" d" e" f" TargetID 2N.AVG_Sig 2N.Det Pval.AVG_Sig.Det Pval Diff Pval DiffScore SYMBOL ILMN_26396 18.35238 0.0080058 44.81118 0.0021834 0.000323 34.90542 KRTHA4 ILMN_15812 439.2499 0 610.0316 0 0.00053 32.75721 SLC2A3 ILMN_22379 778.3962 0 1071.648 0 0.000964 30.16089 PCGF4 ILMN_22043 54.28146 0.0014556 31.45441 0.0029112 0.000966-30.15065 RAB26 ILMN_10954 55.69347 0.0014556 30.69016 0.0029112 0.000901-30.45122 FGF20 ILMN_27201 515.8552 0 377.9779 0 0.000804-30.94495 RGL1 ILMN_11871 275.3153 0 155.1232 0 0.000769-31.14102 AKR1C3 ILMN_10556 185.9658 0 125.8244 0 0.000725-31.39491 SLC16A10 ILMN_2453 225.1933 0 150.8763 0 0.000665-31.77423 TSPAN3 ILMN_21404 86.78853 0.0007278 55.87777 0.0007278 0.000623-32.05733 NOV ILMN_22069 143.8192 0 96.79633 0.0007278 0.000298-35.25298 HIST2H4 ILMN_30616 51.421 0.0021834 23.6715 0.0065502 6.23E-05-42.05549 LOC648517 ILMN_11065 20.88196 0.007278-1.312745 0.6295488 2.62E-05-45.81802 APIN ILMN_7395 514.5203 0 307.792 0 4.75E-06-53.23692 ABCB1 ILMN_7370 230.1354 0 139.9379 0 1.19E-06-59.22792 METTL7B ILMN_138636 148.0692 0 85.58659 0.0007278 1.03E-06-59.85924 CFTR ILMN_17221 42.0515 0.0029112 2.345726 0.2321689 9.45E-07-60.24375 FAM112B ILMN_6950 618.666 0 336.9032 0 4.81E-08-73.17954 SGNE1 ILMN_16602 82.26515 0.0007278 26.86142 0.003639 3.54E-12-114.5157 TDO2! CA! D!>G2! no!dcb! 1.5%! 3.2%! Release!4h! 92.0%! :! Release!2d! 77.2%! 53.8%! Release!3d! 71.0%! :! Release!5d! 59.0%! :! Release!7d! 36.0%! 54.3%!!!Aurora!A!!!CP110! Release!17d! 13.3%! 42.5%! Release!25d! 5.8%! 42.8%! Supplementary Figure 1. Characterisation of DLD1 isogenic cell lines. (a) Comparative Genomic Hybridisation (CGH) analysis of DLD1 cells ( ) relative to a reference D ( ) and relative to early passage (p12) 2N and cells. (b) Cell cycle profile of early (p12; left) and late (p55; right) passage 2N and cells and CGH analysis of 2N and cells. (c) Parental DLD1, 2N, and CA cell growth rates. (d) Gene expression microarray analysis of 2N and cells. (e) Immunocytochemistry images of CA cells demonstrating asymmetrical clustering. (f) Pulse chase of 2N cells following 28h treatment with 5µM DCB; percentage of cells with multiple centrosomes (>2 centrosomes or >2 centrioles/ centrosome) across CA column and percentage of cells with D content higher than diploid G2 across D >G2 column.
1.2 1.2 1 1 2N 0.8 2N 0.6 0.4 SF 0.8 0.4 CA 0.2 0.2 0μ M 10 50 25.5 12 25 12 6. 3. 56 1. SO 78 DM 8 sit OX M OC K d" e" CA siska2 sidnci2!"#$%&$'()"*+,-./0+ 1"'"#2&"+34"%$)53"+ 67('23$'89:+ 1"'"#2&"+)$;-#-)5+ <$73$(%*+ sidncl2a 7 6 5 4 3 2 L 1 TR sic 0 0 sikifc1 CA 0.6 0. SF c" CA a" siapc11 siapc5 siapc10 CA siactr1a siska3 f" Supplementary Figure 2. screening setup and hits. (a) Surviving fraction (SF) of 2N, and CA cells 96h after knocking down KIFC1 expression using 8 different oligos. (b) Surviving fraction (SF) of 2N, and CA cells after treatment with griseofulvin. (c) Immunocytochemistry images of cells treated with the positive control sikifc1. (d) Connectivity analysis of the hits from the screening overlaid by validation results. Coloured boxes indicate whether viability or phenotypic hits are confirmed by deconvolution of pool and/or chemical compound; pool results are shown where deconvolution was not performed. (e,f) Immunocytochemistry images of cells treated with the indicated representative hits. Cells were stained using Aurora A antibody (green) and propidium iodide (red).
a" MDA MB468 SUM149 MDA MB453 T47D MDA MB231 BT549 DMSO!!Eg5!!!Aurora!A! protame ZR75.1 Hs578T MDA MB436 SUM159 MDA MB157 MDA MB361 DMSO protame sicdc2 siprc1 siespl1 CA Supplementary Figure 3. Mitotic spindle morphology in a panel of breast cancer cell lines treated with protame and false positives. (a) Immunocytochemistry images of cells treated with 20 µm protame for 24h. Cells were stained for Aurora A (red), Eg5 (green), and D using DAPI (blue). (b) Immunocytochemistry images or the indicated representative false positive hits. Cells were stained using Aurora A antibody (green) and propidium iodide (red).
a" 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% sictrl CDC20 1 CDC20 2 CDC20 3 CDC20 4 CDC20pool CDC20p/MON5 CDC20p/MON15 CDH1 1 CDH1 2 CDH1 3 CDH1 4 CDH1pool CDH1p/MON5 CDH1p/MON15 2N!!!Eg5!!!Aurora!A! Supplementary Figure 4. Deconvolution of CDC20 pool and effect CDC20/CDH1 depletion in 2N cells. (a) Immunocytochemistry images of 2N cells transfected with the indicated s (pool and individual). pool-transfected cells were additionally treated with the indicated concentrations of monastrol. Cells were stained using specific antibodies against Eg5 (green), Aurora A (red) and DAPI for D (top). Quantification of bipolar, multipolar and monopolar spindles (n = >150 mitoses/ condition; bottom). (b) Immunoblot assays in lysates from sicdc20 transfected cells using the indicated pools or individual s. α-tubulin is used as loading control.
a" ($!"# (!!"# '!"# &!"# %!"# $!"#!"# )*+,-.+-/# 0123# 120% 100% 80% Asynchronous Nocodazole-released MG132-released 012(3# 45# 4560123# 456012(3# 789# 5:;1<# 0=>?@A>.,# 0A*A@A>.,# BC@A>.,# ($!"# (!!"# '!"# &!"# %!"# $!"#!"# )*+,-.+-/# 0123# 012(3# 120% 45# 4560123# 456012(3# 789# 5:;1<# 0=>?@A>.,# 0A*A@A>.,# BC@A>.,# ($!"# (!!"# 100%! CA! 80% '!"# &!"# %!"# $!"#!"# )*+,-.+-/# 0123# 012(3# 45# 4560123# 456012(3# 789# 0:;<=>;.,# 0>*>=>;.,#?@=>;.,# 60% 40% 20% 0% Untreated MON5 MON15 PT PT/MON5 PT/MON15 GSF TAXOL Multipolar Monopolar Bipolar 60% 40% 20% 0% Untreated MON5 MON15 PT PT/MON5 PT/MON15 GSF TAXOL Multipolar Monopolar Bipolar CA c" Overlay CP110 Eg5!!Eg5!!!Aurora!A!!!CP110!!!Eg5! Supplementary Figure 5. Effect of APC/C inhibition at different phases of the cell cycle in spindle multipolarity. (a) Quantification of bipolar, multipolar and monopolar mitoses in cells (corresponding to Fig. 3 a-c; n = >120 mitoses/condition). (b) Immunocytochemistry images of and CA cells after release from thymidine block and treated with the indicated compounds (right); quantification of bipolar, multipolar and monopolar mitoses in and CA cells (n = >120 mitoses/condition). Schematic of the treatment regimen used to assess the effect of the indicated compound treatment on spindle phenotype in and CA cells is shown (bottom). (c) Representative immunocytochemistry images (maximum projections of confocal z-stacks) of CA cells 20 h after release from 4 h MG132 block and treated with protame, experimental conditions are identical as in Figure 3c PT. PT=proTAME, MON=Monastrol
Sor2ng!36h!a7er!transfec2on! 1!week!a7er!sor2ng! ΔCEg5BGFP! wteg5bgfp! GFP!nega2ve! Supplementary Figure 6. FACS sorting of Eg5-GFP populations. FACS sorting of 2N cells 36h after transfection with wteg5-gfp or ΔCEg5-GF at 36h after transfection (left; gate P4) and FACS analysis 7 days later (right). Untransfected 2N cells (GFP negative) are used as controls to determine GFP positive cells and are not sorted
a" WTEg5-GFP ΔCEg5-GFP *!!!!" '$!!!!" )!!!!" '#!!!!" (!!!!" '!!!!!" CTF '!!!!" &!!!!" %!!!!",-./0-0" 1#" CTF &!!!!" %!!!!",-./0-0" 1'" $!!!!" $!!!!" #!!!!" #!!!!"!" #" $" %" &" '" (" )" *" +" #!" ##" #$" #%" #&" #'"!" '" #" (" $" )" %" *" &" +" '!" ''" '#" '(" '$" ')" 1 2 3 1 2 3 Mitosis Not measured 10 15 20 28 29 30 10 15 20 28 29 30 G1 c" CDC27! High! Exposure! Supplementary Figure 7. GFP Fluorescence measurements and in vitro ubiquitination assay. (a) Corrected Total Fluorescence (CTF) measurements from 15 individual cells in mitosis and in the respective daughter cells in G1 in WTEg5-GFP cells and ΔCEg5-GFP cells (top). Representative frames of WTEg5-GFP and ΔCEg5-GFP in mitosis and in G1 (bottom); CTF was measured in frames 1-3 (mitosis) and 28-30 (G1) (b) Low exposure of blot shown in right panel of Fig. 4e. (c) High exposure of CDC27 blot shown in Figure 4d.
a" c" d" e" Supplementary Figure 8. Full scans of Blots blots shown in Figure 2c (a), 4b (b), 4c (c), 4d (d) and 4e (e)
Supplementary"Table"1."An5bodies"used"in"this"work."" Antigen Source Ig Source Dilution Catalogue No. Use Eg5 Mouse Abcam 1:1000 51976 WB Eg5 Mouse Abcam 1:800 51976 IF Eg5 Mouse Santa Cruz 1:500 sc-53691 WB GFP Mouse Abcam 1:2000 ab1218 WB GFP Mouse Abcam 1:2000 ab1218 IF GFP Rabbit Abcam 1:3000 ab6556 IF GFP Rabbit Abcam 2 ug ab6556 IP Aurora A Mouse BD 1:1000 610939 WB Aurora A Mouse BD 1:800 610939 IF CDC27 Mouse Abcam 1:1000 ab10538 WB CDH1 Mouse Abcam 1:1000 ab3242 WB CDC20 Rabbit Abcam 1:1000 ab26483 WB MPM2 Mouse Millipore 1:1000 05-638 WB Tubulin Mouse Sigma 1:10000 T5168 WB FLAG Mouse Clontech 1:5000 635691 WB CP110 Rabbit Gift from E. Nigg 1:1000 - IF