Supporting Information - PNAS · russi–Beadle Ringer’s solution [129 mM NaCl, 4.7 mM KCl, 1.9...
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Supporting Information Moutinho-Pereira et al. 10.1073/pnas.1320013110 SI Materials and Methods Cell Culture, RNAi, and Immunofluorescence. For the primary screen, Drosophila S2 cells were cultured as previously described (1). dsRNA [1 μg, supplemented with 1 μg per well of centrosomin (Cnn) dsRNA and 0.25 μg per well of cell division cycle 27 (Cdc27) dsRNA] was added to the cell culture in 96-well plates. Cdc27 was used to overcome the problem of a low mitotic index typical of S2 cells. At the end of RNAi treatment (day 4), cells were resuspended; transferred to glass-bottomed, Con A-coated, 96/384-well plates (MatriCal Bioscience, Inc.); and allowed to adhere for 2 h before fixation. Cells were fixed in 6.4% (vol/vol) paraformaldehyde for 10 min and incubated overnight at 4 °C with rat anti–α-tubulin YOL1/34 (1:10; Serotec) and rabbit anti- histone H3 phospho-Ser10 (1:1,000; Upstate) antibodies in PBS containing 0.1% Triton and 0.5 mg/mL BSA, followed by im- munostaining with secondary antibodies anti-rat FITC (1:300; Sigma), anti-rabbit Alexa 647 (1:1,000; Invitrogen, Molecular Probes), and DAPI (1 μg/mL). For the entire cell culture and immunostaining procedure, we used the WellMate Automated Pipetting system (Matrix Technologies Corp.). Specimens were imaged in an automated microscope, the ImageXpress Micro (Molecular Devices), using a 40×, 0.95-N.A. PlanApo dry ob- jective lens (Nikon) to achieve high resolution. We imaged three channels (DAPI, FITC, and Cy5) at typically 30–50 sites per well to obtain 200 metaphase cells per well on average. A Polara Robot (Thermo Scientific) was used to place the plates on the micro- scope, especially during overnight acquisitions. For secondary immunofluorescence studies, cells were placed on 0.5 mg/mL Con A (Sigma)-coated coverslips and fixed after 2 h with 6.4% paraformaldehyde in cytoskeleton buffer (2) at room tempera- ture, extracted with 0.5% Triton X-100 in cytoskeleton buffer for 10 min, and incubated overnight at 4 °C with appropriate anti- bodies. The following primary antibodies were used: mouse anti– α-tubulin (mouse clone B512; Sigma), guinea-pig Cnn (1:2,000; a gift from Tim Megraw, Florida State University, Tallahassee, FL), mouse anti-Bj1 (1:100; a gift from M. Frasch, University of Erlangen-Nuremberg, Erlangen, Germany), rabbit antifibrillarin (1:2,000; Abcam), and mouse antihistone H3 phospho-Ser10 (1:100,000; Abcam). Secondary antibodies used were Alexa 488, 568, and 647 (Invitrogen, Molecular Probes) and DAPI (1 μg/ mL). The 3D datasets of representative cells were collected with a Zeiss Imager.Z1 microscope equipped with an AxiocamMRm CCD, using a 100× 1.4-N.A. oil immersion objective. The entire system was driven by Axiovision software. Images were sub- sequently blind-deconvolved with AutoDeblur X2 software (Me- dia Cybernetics) and processed for publication using Adobe Photoshop CS3 and Illustrator 8.0 (Adobe Systems). Microtubule Depolymerization/Repolymerization Assay and Chromo- some Spreads. Microtubules (MTs) were depolymerized at 4 °C for 2 h and allowed to repolymerize at room temperature for different times: 0, 1, 3, and 10 min. Cells were then immediately fixed with methanol at −20 °C for 3 min and processed for im- munofluorescence as described. For chromosome spreads, cells were grown in six-well plates and treated with 30 μM colchicine (Sigma) for ∼16 h; 20 μM MG132 (Sigma) was used for 2 h before fixation. For the Aurora B inhibition assay, 40 μM binu- cleine 2 was added before fixation. Cells were then counted, centrifuged, and resuspended at 10 5 cells per milliliter in Eph- russi–Beadle Ringer’s solution [129 mM NaCl, 4.7 mM KCl, 1.9 mM CaCl 2 , 10 mM Hepes (pH 6.9)] for nonhypotonic treatment while in the presence of both drugs (MG132 and colchicine). A total of 500 μL of the cell suspension was spun onto glass slides at 102.8 × g for 5 min in a Cytospin 2 (Shandon) and immedi- ately fixed and processed for immunofluorescence as described. Primary antibodies used were rabbit anti-Aurora B (1:500; a gift from M. Carmena and B. Earnshaw, University of Edinburgh, Edinburgh, United Kingdom) and rabbit anti-BubR1 and anti- Mad2 as well as rat anti-Cid (1:300, 1:10, and 1:2,000, respec- tively; a gift from C. Sunkel, Instituto de Biologia Molecular e Celular, University of Porto). Screen Image Analysis and Manual Scoring of Spindle Phenotypes. Mitotic cells were automatically identified based on the inten- sity of nuclear anti- phosphohistone H3 staining, and galleries were generated for each gene. A detailed description of nuclei and mitotic cell identification can be found in the supplemental material in ref. 1. Galleries with spindle images from each well were classified visually. The manual classification was performed using the same graphical user interface (GUI) as in ref. 1. This GUI allowed a quick visualization of each gallery and its clas- sification according to different category assignments. The phe- notypic categories were as follows: (i ) few/no spindles, (ii ) weak/ not robust spindles/dim MTs, (iii ) short spindles, (iv) disorga- nized spindles, (v) unfocused spindles, (vi ) long spindles, (vii ) monopolar spindles, (viii ) few mitotic cells, (ix) generally de- fective, and (x) misaligned chromosomes. Specific phenotypes were described by an observer with three ranks: “clear,”“likely,” and “weak” based upon the phenotype clarity and prevalence. In many cases, more than one phenotype was detected for a gene (e.g., “misaligned chromosomes, clear”; “long spindle, likely”). From Initial to Final “Hit” Selection: Repeat RNAi Screening and Minimizing Off-Target Effects. There are two inherent problems commonly associated with this type of screen: nonreproducible phenotypes and off-target effects (3, 4) (discussed in detail in ref. 1). To establish rigorous criteria for minimizing this possibility in our final list of genes, we performed several secondary manual studies and without Cdc27 depletion (to avoid frequent un- specific phenotypes, such as chromosome misalignment). Repeat RNAi experiments were carried out for 191 genes (putative hits from the primary screen) with independently synthesized dsRNA molecules, targeting parts of the gene not targeted in the initial experiment. In most cases, the second, nonoverlapping dsRNA probe was used for confirming the primary hit in the second round of RNAi screening. For repeat dsRNAs, the T7 promoter sequence was directly attached to the primers and the template for in vitro transcription was synthesized in a single round of PCR using genomic DNA as a template. If two different, non- overlapping dsRNAs yielded identical phenotypes, we concluded that the phenotype was not derived from off-target effects. GFP Tagging and Generation of an S2 Cell Line Stably Expressing GFP- Deterin. GFP tagging was performed using the Gateway system (Invitrogen). Full-length cDNA was amplified by PCR using S2 cDNA as a template (Berkeley Drosophila Genome Project clones RH12258, LD23683, AT23067, LD45195, RE55472, RH08789, GH01229, and LD2383). GFP fusion genes (N-terminal and/or C-terminal) were inserted behind the copper-inducible metal- lothionein promoter (pMT vector; Invitrogen) and pBlastDest plasmids (a gift from Mónica Bettencourt-Dias, Instituto Gulben- kian de Ciência, Oeiras, Portugal). Several transient transfections were performed to determine GFP localization, and a cell line stably expressing GFP-deterin was established. GFP localization was Moutinho-Pereira et al. www.pnas.org/cgi/content/short/1320013110 1 of 9
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Supporting InformationMoutinho-Pereira et al. 10.1073/pnas.1320013110SI Materials and MethodsCell Culture, RNAi, and Immunofluorescence.For the primary screen,Drosophila S2 cells were cultured as previously described (1).dsRNA [1 μg, supplemented with 1 μg per well of centrosomin(Cnn) dsRNA and 0.25 μg per well of cell division cycle 27(Cdc27) dsRNA] was added to the cell culture in 96-well plates.Cdc27 was used to overcome the problem of a low mitotic indextypical of S2 cells. At the end of RNAi treatment (day 4), cellswere resuspended; transferred to glass-bottomed, Con A-coated,96/384-well plates (MatriCal Bioscience, Inc.); and allowed toadhere for 2 h before fixation. Cells were fixed in 6.4% (vol/vol)paraformaldehyde for 10 min and incubated overnight at 4 °Cwith rat anti–α-tubulin YOL1/34 (1:10; Serotec) and rabbit anti-histone H3 phospho-Ser10 (1:1,000; Upstate) antibodies in PBScontaining 0.1% Triton and 0.5 mg/mL BSA, followed by im-munostaining with secondary antibodies anti-rat FITC (1:300;Sigma), anti-rabbit Alexa 647 (1:1,000; Invitrogen, MolecularProbes), and DAPI (1 μg/mL). For the entire cell culture andimmunostaining procedure, we used the WellMate AutomatedPipetting system (Matrix Technologies Corp.). Specimens wereimaged in an automated microscope, the ImageXpress Micro(Molecular Devices), using a 40×, 0.95-N.A. PlanApo dry ob-jective lens (Nikon) to achieve high resolution. We imaged threechannels (DAPI, FITC, and Cy5) at typically 30–50 sites per wellto obtain 200 metaphase cells per well on average. A Polara Robot(Thermo Scientific) was used to place the plates on the micro-scope, especially during overnight acquisitions. For secondaryimmunofluorescence studies, cells were placed on 0.5 mg/mLCon A (Sigma)-coated coverslips and fixed after 2 h with 6.4%paraformaldehyde in cytoskeleton buffer (2) at room tempera-ture, extracted with 0.5% Triton X-100 in cytoskeleton buffer for10 min, and incubated overnight at 4 °C with appropriate anti-bodies. The following primary antibodies were used: mouse anti–α-tubulin (mouse clone B512; Sigma), guinea-pig Cnn (1:2,000;a gift from Tim Megraw, Florida State University, Tallahassee,FL), mouse anti-Bj1 (1:100; a gift from M. Frasch, University ofErlangen-Nuremberg, Erlangen, Germany), rabbit antifibrillarin(1:2,000; Abcam), and mouse antihistone H3 phospho-Ser10(1:100,000; Abcam). Secondary antibodies used were Alexa 488,568, and 647 (Invitrogen, Molecular Probes) and DAPI (1 μg/mL). The 3D datasets of representative cells were collected witha Zeiss Imager.Z1 microscope equipped with an AxiocamMRmCCD, using a 100× 1.4-N.A. oil immersion objective. The entiresystem was driven by Axiovision software. Images were sub-sequently blind-deconvolved with AutoDeblur X2 software (Me-dia Cybernetics) and processed for publication using AdobePhotoshop CS3 and Illustrator 8.0 (Adobe Systems).
Microtubule Depolymerization/Repolymerization Assay and Chromo-some Spreads. Microtubules (MTs) were depolymerized at 4 °Cfor 2 h and allowed to repolymerize at room temperature fordifferent times: 0, 1, 3, and 10 min. Cells were then immediatelyfixed with methanol at −20 °C for 3 min and processed for im-munofluorescence as described. For chromosome spreads, cellswere grown in six-well plates and treated with 30 μM colchicine(Sigma) for ∼16 h; 20 μM MG132 (Sigma) was used for 2 hbefore fixation. For the Aurora B inhibition assay, 40 μM binu-cleine 2 was added before fixation. Cells were then counted,centrifuged, and resuspended at 105 cells per milliliter in Eph-russi–Beadle Ringer’s solution [129 mM NaCl, 4.7 mM KCl, 1.9mM CaCl2, 10 mM Hepes (pH 6.9)] for nonhypotonic treatmentwhile in the presence of both drugs (MG132 and colchicine). A
total of 500 μL of the cell suspension was spun onto glass slidesat 102.8 × g for 5 min in a Cytospin 2 (Shandon) and immedi-ately fixed and processed for immunofluorescence as described.Primary antibodies used were rabbit anti-Aurora B (1:500; a giftfrom M. Carmena and B. Earnshaw, University of Edinburgh,Edinburgh, United Kingdom) and rabbit anti-BubR1 and anti-Mad2 as well as rat anti-Cid (1:300, 1:10, and 1:2,000, respec-tively; a gift from C. Sunkel, Instituto de Biologia Molecular eCelular, University of Porto).
Screen Image Analysis and Manual Scoring of Spindle Phenotypes.Mitotic cells were automatically identified based on the inten-sity of nuclear anti- phosphohistone H3 staining, and gallerieswere generated for each gene. A detailed description of nucleiand mitotic cell identification can be found in the supplementalmaterial in ref. 1. Galleries with spindle images from each wellwere classified visually. The manual classification was performedusing the same graphical user interface (GUI) as in ref. 1. ThisGUI allowed a quick visualization of each gallery and its clas-sification according to different category assignments. The phe-notypic categories were as follows: (i) few/no spindles, (ii) weak/not robust spindles/dim MTs, (iii) short spindles, (iv) disorga-nized spindles, (v) unfocused spindles, (vi) long spindles, (vii)monopolar spindles, (viii) few mitotic cells, (ix) generally de-fective, and (x) misaligned chromosomes. Specific phenotypeswere described by an observer with three ranks: “clear,” “likely,”and “weak” based upon the phenotype clarity and prevalence. Inmany cases, more than one phenotype was detected for a gene(e.g., “misaligned chromosomes, clear”; “long spindle, likely”).
From Initial to Final “Hit” Selection: Repeat RNAi Screening andMinimizing Off-Target Effects. There are two inherent problemscommonly associated with this type of screen: nonreproduciblephenotypes and off-target effects (3, 4) (discussed in detail in ref.1). To establish rigorous criteria for minimizing this possibility inour final list of genes, we performed several secondary manualstudies and without Cdc27 depletion (to avoid frequent un-specific phenotypes, such as chromosome misalignment). RepeatRNAi experiments were carried out for 191 genes (putative hitsfrom the primary screen) with independently synthesized dsRNAmolecules, targeting parts of the gene not targeted in the initialexperiment. In most cases, the second, nonoverlapping dsRNAprobe was used for confirming the primary hit in the secondround of RNAi screening. For repeat dsRNAs, the T7 promotersequence was directly attached to the primers and the templatefor in vitro transcription was synthesized in a single round ofPCR using genomic DNA as a template. If two different, non-overlapping dsRNAs yielded identical phenotypes, we concludedthat the phenotype was not derived from off-target effects.
GFP Tagging and Generation of an S2 Cell Line Stably Expressing GFP-Deterin. GFP tagging was performed using the Gateway system(Invitrogen). Full-length cDNA was amplified by PCR using S2cDNA as a template (Berkeley Drosophila Genome Project clonesRH12258, LD23683, AT23067, LD45195, RE55472, RH08789,GH01229, and LD2383). GFP fusion genes (N-terminal and/orC-terminal) were inserted behind the copper-inducible metal-lothionein promoter (pMT vector; Invitrogen) and pBlastDestplasmids (a gift from Mónica Bettencourt-Dias, Instituto Gulben-kian de Ciência, Oeiras, Portugal). Several transient transfectionswere performed to determine GFP localization, and a cell linestably expressingGFP-deterinwas established.GFP localizationwas
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observed after CuSO4 addition to the 500 μM cell culture mediumfor 24 h to induce gene expression.
Live Cell Imaging. S2 cells stably expressing H2B-GFP and mCherry–α-tubulin (1) were plated in 0.25 mg/mL Con A-coated MatTekdishes (MatTek Corporation). The 4D datasets were collectedwith an Andor Revolution Spinning Disk confocal system (An-dor Technology) equipped with an Electron Multiplying CCDiXonEM+ camera and a Yokogawa CSU-22 U (Yokogawa Elec-tric) unit based on an Olympus IX81 inverted microscope. Twolaser lines (488 and 561 nm) were used for near-simultaneousexcitation of GFP and mCherry, and the system was driven byAndor IQ software (Andor Technology). Time-lapse imagestacks were collected every 30 s, for 1 or ∼5 min (in the case ofmultipoint acquisition, the minimal interval is automatically re-calculated), with 0.8-μm z-steps and projected as maximum pixelintensities. For Aurora B inhibition, medium was supplementedwith 40 μM binucleine 2 (Sigma), and for the depolymerizationof MTs, 200 μM colchicine was used.
FRET Measurements. To investigate the formation of a Ras-relatednuclear protein in the GTP-bound state (RanGTP) gradient andassociated cargo, we used the previously developed FRET bio-sensor Rango-2 (5, 6). For analysis of the RanGTP gradient inHeLa cells, they were transiently transfected with X-trem-eGENE (Roche) according to the manufacturer’s instructions.For analysis of RanGTP gradient in S2 cells, we cloned theRango-2 biosensor in a pAc5.1/V5-His vector (Invitrogen) con-taining a blasticidin resistance cassette, using ApaI cloning sites.
This was used to transfect and select S2 cells stably expressingRango-2 using Effectene (Qiagen). For imaging, S2 cells stablyexpressing Rango-2 with and without regulator of chromosomecondensation 1 (RCC1) dsRNA for 96 h were plated in pre-coated MatTek dishes with 0.25 mg/mL Con A and recorded ina Leica TCS SP5 II confocal microscope with an HC PL APO CS40× 1.10 CORR water lens (Leica). In both cell types, cyanfluorescent protein optimized for FRET with YPet (CyPet) wasexcited with the argon laser at 458 nm and CyPet and FRETemission signals were acquired simultaneously at 468–513 nm and525–572 nm, respectively. The acquired images were subtractedfrom the background, and 32-bit ratio images were created withthe Ratio Plus plug-in of ImageJ software (National Institutes ofHealth; http://rsbweb.nih.gov/ij/plugins/ratio-plus.html).
Western Blotting. Protein extracts were boiled for 5 min in SDSsample buffer and subjected to SDS/PAGE, followed by trans-ference to nitrocellulose membranes and Western blot analysiswith the appropriate antibodies. Primary antibodies used weremouse α-tubulin (1:3,000, clone B512; Sigma), rabbit Cnn(1:10,000; a gift from Tim Megraw, Florida State University,Tallahassee, FL), mouse Bj1 (1:100; a gift from Manfred Frasch,University of Erlangen-Nuremberg, Erlangen, Germany), and in-house–generated rabbit GFP. Appropriate secondary antibodieswere detected using ECL (Pierce).
Statistical Analysis. All statistical analyses were performed withGraphPad Prism V5 (GraphPad Software, Inc.). Values were con-sidered statistically different whenever P < 0.05.
1. Goshima G, et al. (2007) Genes required for mitotic spindle assembly in Drosophila S2cells. Science 316(5823):417–421.
2. Maiato H, et al. (2006) The ultrastructure of the kinetochore and kinetochore fiber inDrosophila somatic cells. Chromosoma 115(6):469–480.
3. Ma Y, Creanga A, Lum L, Beachy PA (2006) Prevalence of off-target effects inDrosophila RNA interference screens. Nature 443(7109):359–363.
4. Kulkarni MM, et al. (2006) Evidence of off-target effects associated with long dsRNAsin Drosophila melanogaster cell-based assays. Nat Methods 3(10):833–838.
5. O’Connell CB, Loncarek J, Kaláb P, Khodjakov A (2009) Relative contributions ofchromatin and kinetochores to mitotic spindle assembly. J Cell Biol 187(1):43–51.
6. Kaláb P, Soderholm J (2010) The design of Förster (fluorescence) resonance energytransfer (FRET)-based molecular sensors for Ran GTPase. Methods 51(2):220–232.
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Fig. S1. Characterization of spindle assembly and respective dependence on centrosomes and RanGTP in Drosophila S2 cells. (A) Western blot analysis of RCC1and/or Cnn depletions by RNAi. α-Tubulin was used as a loading control. (B) Quantification of nuclear envelope breakdown (NEB) to anaphase duration incontrol and RCC1 + Cnn RNAi cells represented as whisker plots (n = 10 cells per condition; P > 0.05, ANOVA). The line in the middle of the boxes is plotted atthe median, and “+” represents the mean. (C) Live imaging of S2 cells stably expressing H2B-GFP (red) and mCherry–α-tubulin (green) upon RCC1 + Cnn RNAi.Time is expressed as minutes:seconds. (Scale bar: 5 μm.)
Fig. S2. Identification of new proteins required for normal spindle assembly. (A) Depletion of Cnn (Cnn−) together with either NS1, Non1, Non2, Non3, Ssp6,Ssp7, or Vps 33B led to a pronounced short metaphase spindle phenotype. The short-spindle phenotype is also evident when centrosomes are present (Cnn+).α-Tubulin is shown in green, DNA is shown in blue, and Cnn is shown in red. (B) Quantification of metaphase spindle length (distance between minus ends) incells containing centrosomes, represented as whisker plots (n = 50 cells per condition). All RNAi conditions showed shorter spindles relative to controls (*P <0.05, Dunnett’s test). The line in the middle of the boxes is plotted at the median, and “+” represents the mean. (C–F) S2 cells transiently transfected with NS1-GFP, GFP-Non1, Non2-GFP, or GFP-Non3 (green) show a specific localization in the nucleolus, as judged by colocalization with fibrillarin (red). (Scale bars: 5 μm.)
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Fig. S3. Effect of deterin RNAi or Aurora B inhibition on acentrosomal spindle assembly. (A) Western blotting shows the depletion of GFP-deterin after Cnn +deterin RNAi; α-tubulin was used as a loading control. (B) Quantification of metaphase spindle length in different experimental conditions, represented aswhisker plots (*P < 0.001, Mann–Whitney test). The line in the middle of the boxes is plotted at the median, and “+” represents the mean. The long astral MTstypical of cells depleted of Klp10A in the presence of centrosomes might counteract centrosome-independent spindle pushing forces that ultimately help in
Legend continued on following page
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determining metaphase spindle length. (C and D) After Cnn RNAi, Aurora B (red) is found within the inner centromere region, as expected in control cells, butwas lost from centromeres upon depletion of both Cnn and deterin. (E) Quantification of prometaphase and metaphase cells in Cnn RNAi cells vs. Cnn RNAi +binucleine 2 after proteasome inhibition with MG132. (F) Quantification of bipolarity and spindle defects after Cnn RNAi, Cnn RNAi + binucleine 2, and Cnn +RCC1 RNAi + binucleine 2 in the presence of MG132. Bars represent the mean of three independent experiments (n =100 cells per condition per experiment).Error bars represent SEM. (G and H) Representative images illustrate data from E. DNA is shown in blue, phosphohistone H3 on S10 is shown in red, andα-tubulin is shown in green. (Scale bars: 5 μm.)
Fig. S4. MT repolymerization and mitotic spindle reassembly with/without functional centrosomes is unaffected by RCC1 depletion. (A and B) Efficiency of MTrepolymerization and spindle assembly in S2 cells at different time points collected after cold-induced depolymerization in the absence of Cnn and/or RCC1 (n =20 cells per condition). DNA (blue), α-tubulin (green,) and RCC1 (red) are shown. (Scale bars: 5 μm.) (C) Quantitative Western blot analysis of RCC1 depletionefficiency in S2-U. α-Tubulin was used as a loading control.
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Movie S1. Live imaging of chromosomes (H2B-GFP) and MTs (mCherry–α-tubulin) during spindle assembly in a control S2 cell. The time lapse is 30 s. Controlcells have two centrosomes that nucleate astral MTs that will participate in spindle assembly.
Movie S1
Movie S2. Live imaging of chromosomes (H2B-GFP) and MTs (mCherry–α-tubulin) during spindle assembly after Cnn RNAi. The time lapse is 30 s. Note thatMTs organize around chromatin and the vicinity of the nuclear envelope.
Movie S2
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Movie S3. Live imaging of GFP-deterin in an S2 cell. The time lapse is 30 s. GFP-deterin decorates the inner centromere from prophase/prometaphase untilearly anaphase, transferring to the spindle midzone in late anaphase and telophase.
Movie S3
Movie S4. Live imaging of chromosomes (H2B-GFP) and MTs (mCherry–α-tubulin) during spindle assembly after codepletion of deterin and Cnn. The timelapse is 30 s. After knocking down deterin and Cnn, ∼50% of cells assemble a bipolar spindle, but they enter an anaphase-like stage with their chromatinprematurely decondensed and fail to segregate their chromosomes correctly.
Movie S4
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Movie S5. Live imaging of chromosomes (H2B-GFP) and MTs (mCherry–α-tubulin) during spindle assembly after Cnn RNAi and addition of 40 μM binucleine 2.The time lapse is 30 s. After addition of binucleine 2 to Cnn RNAi cells, a bipolar spindle forms in 60% of the cells, but they fail to segregate their chromosomescorrectly because DNA decondenses prematurely, similar to deterin depletion.
Movie S5
Movie S6. Live imaging of chromosomes (H2B-GFP) and MTs (mCherry–α-tubulin) during spindle assembly after double RNAi of Cnn and CDC27 and afteraddition of 40 μM binucleine 2. The time lapse is 30 s. After addition of binucleine 2 to Cnn + CDC27 RNAi cells, a bipolar spindle forms and chromosomes arekept condensed. Note that chromosome congression is delayed, likely due to an inability to correct erroneous kinetochore–MT attachments.
Movie S6
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Movie S7. Live imaging of chromosomes (H2B-GFP) and MTs (mCherry–α-tubulin) during spindle assembly after depletion of Cnn and Bj1/RCC1. The time lapseis 30 s.
Movie S7
Table S1. List of target sequences from potential uncharacterized/unexpected genes identified in our first automated screen
Table S1
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