a" b" 2N c" d" e" f" !!Aurora!A!!!CP110!
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1 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_ KRTHA4 ILMN_ SLC2A3 ILMN_ PCGF4 ILMN_ RAB26 ILMN_ FGF20 ILMN_ RGL1 ILMN_ AKR1C3 ILMN_ SLC16A10 ILMN_ TSPAN3 ILMN_ NOV ILMN_ HIST2H4 ILMN_ E LOC ILMN_ E APIN ILMN_ E ABCB1 ILMN_ E METTL7B ILMN_ E CFTR ILMN_ E FAM112B ILMN_ E SGNE1 ILMN_ E 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.
2 N 0.8 2N SF CA μ M 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 L 1 TR sic 0 0 sikifc1 CA 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).
3 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).
4 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.
5 a" ($!"# (!!"# '!"# &!"# %!"# $!"#!"# )*+,-.+-/# 0123# 120% 100% 80% Asynchronous Nocodazole-released MG132-released 012(3# 45# # (3# 789# 5:;1<# ($!"# (!!"# '!"# &!"# %!"# $!"#!"# )*+,-.+-/# 0123# 012(3# 120% 45# # (3# 789# 5:;1<# ($!"# (!!"# 100%! CA! 80% '!"# &!"# %!"# $!"#!"# )*+,-.+-/# 0123# 012(3# 45# # (3# 789# 0:;<=>;.,# 60% 40% 20% 0% Untreated MON5 MON15 PT PT/MON5 PT/MON15 GSF TAXOL Multipolar Monopolar Bipolar 60% 40% 20% 0% Untreated MON5 MON15 PT PT/MON5 PT/MON15 GSF TAXOL Multipolar Monopolar Bipolar CA c" Overlay CP110 Eg5!!Eg5!!!Aurora!A!!!CP110!!!Eg5! Supplementary Figure 5. Effect of APC/C inhibition at different phases of the cell cycle in spindle multipolarity. (a) Quantification of bipolar, multipolar and monopolar mitoses in cells (corresponding to Fig. 3 a-c; n = >120 mitoses/condition). (b) Immunocytochemistry images of and CA cells after release from thymidine block and treated with the indicated compounds (right); quantification of bipolar, multipolar and monopolar mitoses in and CA cells (n = >120 mitoses/condition). Schematic of the treatment regimen used to assess the effect of the indicated compound treatment on spindle phenotype in and CA cells is shown (bottom). (c) Representative immunocytochemistry images (maximum projections of confocal z-stacks) of CA cells 20 h after release from 4 h MG132 block and treated with protame, experimental conditions are identical as in Figure 3c PT. PT=proTAME, MON=Monastrol
6 Sor2ng!36h!a7er!transfec2on! 1!week!a7er!sor2ng! ΔCEg5BGFP! wteg5bgfp! GFP!nega2ve! Supplementary Figure 6. FACS sorting of Eg5-GFP populations. FACS sorting of 2N cells 36h after transfection with wteg5-gfp or ΔCEg5-GF at 36h after transfection (left; gate P4) and FACS analysis 7 days later (right). Untransfected 2N cells (GFP negative) are used as controls to determine GFP positive cells and are not sorted
7 a" WTEg5-GFP ΔCEg5-GFP *!!!!" '$!!!!" )!!!!" '#!!!!" (!!!!" '!!!!!" CTF '!!!!" &!!!!" %!!!!",-./0-0" 1#" CTF &!!!!" %!!!!",-./0-0" 1'" $!!!!" $!!!!" #!!!!" #!!!!"!" #" $" %" &" '" (" )" *" +" #!" ##" #$" #%" #&" #'"!" '" #" (" $" )" %" *" &" +" '!" ''" '#" '(" '$" ')" Mitosis Not measured 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 (G1) (b) Low exposure of blot shown in right panel of Fig. 4e. (c) High exposure of CDC27 blot shown in Figure 4d.
8 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)
9 Supplementary"Table"1."An5bodies"used"in"this"work."" Antigen Source Ig Source Dilution Catalogue No. Use Eg5 Mouse Abcam 1: WB Eg5 Mouse Abcam 1: IF Eg5 Mouse Santa Cruz 1:500 sc 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: WB Aurora A Mouse BD 1: 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: WB Tubulin Mouse Sigma 1:10000 T5168 WB FLAG Mouse Clontech 1: WB CP110 Rabbit Gift from E. Nigg 1: IF
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DOI: 10.1038/ncb3076 Supplementary Figure 1 btrcp targets Cep68 for degradation during mitosis. a) Cep68 immunofluorescence in interphase and metaphase. U-2OS cells were transfected with control sirna
Figure S1. HP1α localizes to centromeres in mitosis and interacts with INCENP. (A&B) HeLa
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DOI: 1.138/ncb222 / b. WB anti- WB anti- ulin Mitotic index (%) 14 1 6 2 T (h) 32 48-1 1 2 3 4 6-1 4 16 22 28 3 33 e. 6 4 2 Time (min) 1-6- 11-1 > 1 % cells Figure S1 depletion leads to mitotic defects
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Supplementary Figure S1
Supplementary Figure S1 Supplementary Figure S1. PARP localization patterns using GFP-PARP and PARP-specific antibody libraries GFP-PARP localization in non-fixed (A) and formaldehyde fixed (B) GFP-PARPx
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Supporting Information
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TABLE OF CONTENTS SUPPLEMENTARY LEGENDS... 2 11 MOVIE S1... 2 FIGURE S1 LEGEND... 3 FIGURE S2 LEGEND... 4 FIGURE S3 LEGEND... 5 FIGURE S4 LEGEND... 6 FIGURE S5 LEGEND... 7 FIGURE S6 LEGEND... 8 FIGURE
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DOI: 10.1038/ncb2566 Figure S1 CDKL5 protein expression pattern and localization in mouse brain. (a) Multiple-tissue western blot from a postnatal day (P) 21 mouse probed with an antibody against CDKL5.
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures
Type of file: PDF Title of file for HTML: Supplementary Information Description: Supplementary Figures Type of file: MOV Title of file for HTML: Supplementary Movie 1 Description: NLRP3 is moving along
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SUPPLEENTRY INFORTION DOI: 1.138/ncb2577 Early Telophase Late Telophase B icrotubules within the ICB (percent of total cells in telophase) D G ultinucleate cells (% total) 8 6 4 2 2 15 1 5 T without gaps
Supplementary Information
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sirna pool: Control Tetherin -HA-GFP HA-Tetherin -Tubulin Supplementary Figure S1. Knockdown of HA-tagged tetherin expression by tetherin specific sirnas. HeLa cells were cotransfected with plasmids expressing
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Supplementary Figure 1 AAV-GFP injection in the MEC of the mouse brain C57Bl/6 mice at 4 months of age were injected with AAV-GFP into the MEC and sacrificed at 7 days post injection (dpi). (a) Brains
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Supplementary information
Supplementary information Exosomes mediate the cell-to-cell transmission of interferon alpha-induced antiviral activity Jianhua Li, Kuancheng Liu, Yang Liu, Yan Xu, Fei Zhang, Huijuan Yang, Jiangxia Liu,
p = formed with HCI-001 p = Relative # of blood vessels that formed with HCI-002 Control Bevacizumab + 17AAG Bevacizumab 17AAG
A.. Relative # of ECs associated with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 ol b p < 0.001 Relative # of blood vessels that formed with HCI-001 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 l b p = 0.002 Control IHC:
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Supplementary Figure 1
Supplementary Figure 1 Supplementary Figure 1. Neither the activation nor suppression of the MAPK pathway affects the ASK1/Vif interaction. (a, b) HEK293 cells were cotransfected with plasmids encoding
Supplementary Table; Supplementary Figures and legends S1-S21; Supplementary Materials and Methods
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Supplementary Fig. 1 a CQ treatment ScrB OGX11 MG132 I II AZD5363 I II b GFP / / GFP / / GFP / / GFP / / GFP GFP Actin Actin ctrl CQ GFP / / GFP / / GFP / / GFP / / GFP GFP Actin Actin rapamycin rapamycincq
marker. DAPI labels nuclei. Flies were 20 days old. Scale bar is 5 µm. Ctrl is
Supplementary Figure 1. (a) Nos is detected in glial cells in both control and GFAP R79H transgenic flies (arrows), but not in deletion mutant Nos Δ15 animals. Repo is a glial cell marker. DAPI labels
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Cesarini et al., http ://www.jcb.org /cgi /content /full /jcb /DC1
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Tel.: +98 216 696 9291; Fax: +98 216 696 9291; E-mail: mrasadeghi@pasteur.ac.ir Tel: +98 916 113 7679; Fax: +98 613 333 6380; E-mail: abakhshi_e@ajums.ac.ir A Soluble Chromatin-bound MOI 0 1 5 0 1 5 HDAC2
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DOI: 10.1038/ncb2988 Supplementary Figure 1 Kif7 L130P encodes a stable protein that does not localize to cilia tips. (a) Immunoblot with KIF7 antibody in cell lysates of wild-type, Kif7 L130P and Kif7
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doi:10.1038/nature12652 Supplementary Figure 1. PRDM16 interacts with endogenous EHMT1 in brown adipocytes. Immunoprecipitation of PRDM16 complex by flag antibody (M2) followed by Western blot analysis
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Supplementary Figures Supplementary Figure 1 Characterization of stable expression of GlucB and sshbira in the CT26 cell line (a) Live cell imaging of stable CT26 cells expressing green fluorescent protein
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