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LETTERS

SCFCyclin F controls centrosome homeostasis andmitotic fidelity through CP110 degradationVincenzo D’Angiolella1, Valerio Donato1, Sangeetha Vijayakumar1, Anita Saraf2, Laurence Florens2,Michael P. Washburn2,3, Brian Dynlacht1 & Michele Pagano1,4

Generally, F-box proteins are the substrate recognition subunits ofSCF (Skp1–Cul1–F-box protein) ubiquitin ligase complexes, whichmediate the timely proteolysis of important eukaryotic regulatoryproteins1,2. Mammalian genomes encode roughly 70 F-box pro-teins, but only a handful have established functions3,4. The F-boxprotein family obtained its name from Cyclin F (also called Fbxo1),in which the F-box motif (the 40-amino-acid domain required forbinding to Skp1) was first described5. Cyclin F, which is encoded byan essential gene, also contains a cyclin box domain, but in contrastto most cyclins, it does not bind or activate any cyclin-dependentkinases (CDKs)5–7. However, like other cyclins, Cyclin F oscillatesduring the cell cycle, with protein levels peaking in G2. Despite itsessential nature and status as the founding member of the F-boxprotein family, Cyclin F remains an orphan protein, whose func-tions are unknown. Starting from an unbiased screen, we identifiedCP110, a protein that is essential for centrosome duplication, as aninteractor and substrate of Cyclin F. Using a mode of substratebinding distinct from other F-box protein–substrate pairs, CP110and Cyclin F physically associate on the centrioles during the G2phase of the cell cycle, and CP110 is ubiquitylated by the SCFCyclin F

ubiquitin ligase complex, leading to its degradation. siRNA-mediated depletion of Cyclin F in G2 induces centrosomal andmitotic abnormalities, such as multipolar spindles and asymmetric,bipolar spindles with lagging chromosomes. These phenotypeswere reverted by co-silencing CP110 and were recapitulated byexpressing a stable mutant of CP110 that cannot bind Cyclin F.Finally, expression of a stable CP110 mutant in cultured cells alsopromotes the formation of micronuclei, a hallmark of chromosomeinstability. We propose that SCFCyclin F-mediated degradation ofCP110 is required for the fidelity of mitosis and genome integrity.

To identify substrates of the SCFCyclin F ubiquitin ligase, we transi-ently expressed FLAG-HA-tagged Cyclin F in cells from the human celllines HeLa or HEK-293T and analysed the Cyclin F complex usingimmunopurification with Multidimensional Protein IdentificationTechnology (MudPIT)8. In both cases, MudPIT revealed the presenceof peptides corresponding to Skp1 and Cul1, whereas, in agreementwith previous reports6,9, no peptides corresponding to CDKs wereidentified (Supplementary Fig. 1a). Instead, MudPIT revealed the pres-ence of peptides derived from CP110 (Supplementary Table 1). Bycombining both analyses, we identified 21 spectra, corresponding to12 unique CP110 peptides. In two additional experiments, we immu-nopurified a Cyclin F mutant lacking the cyclin box (Cyclin F(1–270)),and although Skp1 and Cul1 still co-immunoprecipitated with CyclinF(1–270), CP110 was not present (Supplementary Table 1).

CP110 localizes to the distal ends of the centrioles, and its deple-tion interferes with centrosome re-duplication generated either by

arresting cells in S phase for prolonged time periods or by overex-pressing Plk4 (refs 10,11), indicating that CP110 has a pivotal role inthe formation of new centrioles. Similarly, the fly orthologue ofCP110 is necessary for both centriole duplication and centrosomematuration12. Finally, CP110 has additional roles in the regulation ofcentriole length and cilium formation13,14.

To investigate whether the binding between CP110 and Cyclin F isspecific, we expressed in HEK-293T cells fourteen F-box proteins thatwere then immunoprecipitated to evaluate their interaction withCP110. We found that the only F-box protein able to co-immuno-precipitate endogenous CP110 was Cyclin F (Supplementary Fig. 1b).Using synchronized HeLa cells, we found that endogenous Cyclin Fand endogenous CP110 interacted exclusively in the G2 and Mphases, as monitored by immunoblotting for cell cycle markers andflow cytometry (Fig. 1a and data not shown). Subsequently, we mappedthe Cyclin F binding motif of CP110. A series of binding experiments,using multiple CP110 deletion mutants, narrowed the binding motif toa region of human CP110 located between amino acids 565 and 620(Supplementary Fig. 2). This region contains one putative RxL motif,an established cyclin binding domain. A CP110 with a mutation in thismotif (CP110(RxL/AxA)) failed to co-immunoprecipitate endogenousCyclin F (Supplementary Fig. 2a), indicating that the RxL motif,located at residues 588–590, mediates binding to Cyclin F. Cyclinsrecruit RxL-containing proteins through a hydrophobic patch inthe cyclin box domain15. Cyclin F requires its cyclin box to bind endo-genous CP110 (Supplementary Fig. 3a, b and Supplementary Table 1).Moreover, Cyclin F has a conserved hydrophobic patch and a Cyclin Fwith a double mutation in this domain (Cyclin F(M/A;L/A)) lost theability to bind CP110 (Supplementary Fig. 3c, d).

Because of the centrosomal localization of CP110, we investigatedthe subcellular localization of Cyclin F. We co-stained synchronizedU-2OS cells (human osteosarcoma cell line) with antibodies to CyclinF and c-tubulin (a centrosomal marker). As expected, cells in both Sand G2 phase showed two c-tubulin dots, which were distinct (butadjacent) in S phase and separated by at least 2mm in G2. We foundthat, similar to CP110 (refs 10,11.), endogenous Cyclin F partiallycolocalized with c-tubulin (Fig. 1b); however, unlike CP110, whichis exclusively centrosomal (refs 10,11, Fig. 1c and Supplementary Fig.4), Cyclin F also localized to the nucleus. Moreover, Cyclin F stainingon the centrosomes was rare in S phase cells and increased in G2 cells,whereas the intensity of CP110 staining was predominant in S phaseand decreased in G2 phase (Fig. 1b and Supplementary Fig. 4), cor-relating with the total CP110 levels detected by immunoblotting. Wealso observed that Cherry–Cyclin F colocalized with GFP–CP110 andwas present on both the mother and daughter centrioles (Fig. 1c).Finally, we found that the centrosomal localization of Cyclin F does

1Department of Pathology, NYU Cancer Institute, New York University School of Medicine, 522 First Avenue, SRB 1107, New York, New York 10016, USA. 2The Stowers Institute forMedical Research, 1000 East 50th Street, Kansas City, Missouri 64110, USA. 3Department of Pathology and Laboratory Medicine, The University of Kansas Medical Center, 3901Rainbow Boulevard, Kansas City, Kansas 66160, USA. 4Howard Hughes Medical Institute.

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not require its binding to CP110 and is present at the amino terminus,as Cyclin F(1–270) and Cyclin F(M/A;L/A) (neither of which interactswith CP110) still localized to the centrioles, and Cyclin F(271–786)(which contains the CP110-binding domain) completely lost its cen-trosomal localization and binding to CP110 (Supplementary Fig. 3aand data not shown).

As part of our investigation of CP110 binding to Cyclin F, wenoticed that, compared to wild-type Cyclin F, the Cyclin F(LP/AA)mutant (in which the first two amino acids of the F-box domain weremutated to alanine) bound less Skp1 and Cul1 (as expected) butmore CP110 (Supplementary Fig. 3b). This result indicated thatCP110 might be targeted for proteolysis by Cyclin F, becauseCyclin F(LP/AA) cannot form an active ubiquitin ligase; it cansequester CP110 in a more stable complex that is easier to detect.

In agreement with this interpretation, expression of wild-type CyclinF resulted in a marked reduction in endogenous CP110 levels in fourdifferent cell lines. Moreover, expression of either Cyclin F(LP/AA)or Cyclin F(M/A;L/A) had no effect on CP110 levels (Fig. 2a andSupplementary Fig. 5).

To test further whether Cyclin F might regulate the degradation ofCP110, we used three different short interfering RNA (siRNA) oli-gonucleotides to reduce the expression of Cyclin F in synchronizedHeLa cells. We also silenced Cyclin F expression in synchronizedU-2OS and RPE1–hTERT cells (retinal pigmented epithelium cellsimmortalized with hTERT (human telomerase reverse transcrip-tase)) using the most effective of the three oligos. In all cases, deple-tion of Cyclin F inhibited the G2-specific degradation of CP110(Fig. 2b and Supplementary Fig. 6). Finally, immunopurified wild-type Cyclin F, but not Cyclin F(LP/AA), promoted the in vitro ubi-quitylation of CP110 (Fig. 2c).

Together, the results in Figs 1 and 2 and Supplementary Figs 1–6show that Cyclin F mediates the degradation of CP110 in G2 phase byforming an active SCF ubiquitin ligase complex.

To study the biological significance of Cyclin F-mediated degra-dation of CP110, we investigated whether overexpression of Cyclin Fwould affect centrosome reduplication in cells in which DNA syn-thesis is inhibited by hydroxyurea16. Therefore, we forced the express-ion of Cyclin F in U-2OS cells that were subsequently treated for 48 hwith hydroxyurea, inducing a block in S phase (when endogenousprotein levels are low (Supplementary Fig. 7) and Cyclin F is rarelylocalized to the centrosome (Fig. 1b)). Overexpression of Cyclin Finduced its temporal mislocalization to the centrosome (not shown).Cells were analysed for centrosome number by dual-colour, indirect

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Figure 1 | Cyclin F and CP110 interact and colocalize to the centrosomes.a, HeLa cells were synchronized at G1/S using a double-thymidine blockbefore release into fresh medium. Cells were collected at the indicated times,lysed, immunoprecipitated with anti-Cyclin F antibody and immunoblottedas indicated. Last lane shows immunoprecipitation (IP) pre-incubated withthe antigenic peptide. Left panel shows 10% of the material used forimmunoprecipitations (input). b, U-2OS cells were synchronized as ina, fixed, and incubated with an anti-Cyclin F antibody (red) and anti-c-tubulin antibody (green). The third panel shows staining after pre-incubation with the antigenic peptide. DNA was stained with DAPI. Insetsshow magnified views of the centrosomes indicated by arrows. Scalebar, 10mM. The table shows the percentage of cells with centrosomal CyclinF (where 100% was the total cells staining positive for nuclear Cyclin F) atdifferent times after the release from the double-thymidine block (n < 50 pertime point). c, Cyclin F and CP110 colocalize to the centrioles. U-2OS cellswere transfected with GFP-CP110 and Cherry-Cyclin F. In merged images,yellow shows colocalization of CP110 and Cyclin F.

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Figure 2 | CP110 is targeted for ubiquitylation and degradation by SCFCyclin F

during the G2 phase of the cell cycle. a, U-2OS cells were transfected withan empty vector (EV), FLAG-tagged wild-type (WT) Cyclin F, or FLAG-tagged Cyclin F(LP/AA). Twenty-four hours after transfection, cells werecollected, lysed, and immunoblotted as indicated. DNA content wasmonitored by flow cytometry. b, HeLa cells were transfected with siRNAs toeither LacZ or Cyclin F (oligo #2). Cells synchronized as in Fig. 1a were thencollected at the indicated times, lysed and immunoblotted as indicated.DNA content was monitored by flow cytometry. c, HEK-293T cells weretransfected as in a, lysed, immunoprecipitated with anti-FLAG resin, andused in a ubiquitylation assay.

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immunofluorescence using an antibody to c-tubulin and an antibodyto Centrin 2 (a centriole marker). Control U-2OS cells that had beenarrested in S phase underwent centrosome overduplication as deter-mined by the presence of more than two c-tubulin foci and morethan four Centrin 2 foci. By contrast, cells expressing high levels ofCyclin F did not show centrosome overduplication. Importantly, co-expression of a CP110 mutant that cannot bind Cyclin F reverted theCyclin F-mediated inhibition of centrosome overduplication(Supplementary Fig. 7), indicating that Cyclin F acts as an inhibitorof centrosome reduplication by restraining the expression of CP110.

In a complementary approach, we analysed synchronized andsiRNA-treated U-2OS cells for centrosome defects. At both 9 and12 h after release from a G1/S block (when most cells were in G2and early M phases, respectively), treatment with siRNAs targetingCyclin F produced a significant increase in the percentage of cellsshowing more than two c-tubulin foci and more than four foci forboth Centrin 2 and CP110 (Fig. 3a and Supplementary Fig. 8).Significantly, expression of a wild-type, but siRNA-insensitive,Cyclin F rescued the induction of excess CP110 dots. However,expression of the siRNA-resistant Cyclin F(M/A;L/A) failed to rescuethe phenotype (Supplementary Fig. 8). Finally, in agreement with thesiRNA results, we observed that Cyclin F2/2 mouse embryonic fibro-blasts (MEFs) showed CP110 accumulation and an increased numberof c-tubulin and Centrin 2 foci compared with Cyclin FFlox/– MEFs(Supplementary Fig. 9).

During mitosis, the assembly of a bipolar spindle is mediated bythe centrosomes (from which microtubules nucleate) and ensures theproper segregation of the genetic material. Cells react to centrosomeoverduplication by centrosome clustering, to prevent the formationof multipolar spindles17–19. However, before centrosome clustering,cells with extra centrosomes pass through transient, multipolar inter-mediates that promote merotelic and syntelic attachments, with con-sequent lagging chromosomes20. Therefore, we analysed mitoticfigures using an antibody to a-tubulin (to stain mitotic spindles),an antibody to Centrin 2 and 49,6-diamidino-2-phenylindole(DAPI). Mitotic cells in which Cyclin F levels were reduced showed

an increase in both multipolar spindles and asymmetric, bipolarspindles with lagging chromosomes (Fig. 3b and SupplementaryFig. 10). Significantly, when CP110 was silenced together withCyclin F, the number of Centrin 2/c-tubulin foci and mitotic aberra-tions reverted to approximately control levels (Fig. 3a, b andSupplementary Figs 10, 11), strongly indicating that the accumula-tion of CP110 during G2 phase in Cyclin F-depleted cells is respons-ible for the observed phenotypes.

To further investigate the effects produced by the failure to degradeCP110 in G2, we analysed synchronized U-2OS cells expressing eitherFLAG-tagged wild-type CP110 or FLAG-tagged CP110(RxL/AxA).Wild-type CP110 was degraded in G2 and M phase, whereasCP110(RxL/AxA), consistent with its inability to bind Cyclin F(Supplementary Fig. 2), was stable (Fig. 4a). Importantly, expressionof the stable CP110 mutant recapitulated the centrosome phenotypesobserved upon Cyclin F silencing, namely an increased number offoci positive for Centrin 2, CP110 and c-tubulin (Fig. 4b). Moreover,100% of the mitotic cells that stained positive for CP110(RxL/AxA)showed abnormal spindles and lagging chromosomes (n 5 29/29),whereas only 25% of mitotic cells that did not express CP110(RxL/AxA) showed mitotic aberrations (n 5 7/28) (Fig. 4c). Finally,expression of CP110(RxL/AxA) promoted the formation of micro-nuclei, a hallmark of chromosomal instability (Fig. 4d).

Chromosome instability can result from centrosome overduplica-tion18–20. Here, we show that a defect in the SCFCyclin F-mediateddegradation of CP110 results in centrosome and chromosome aber-rations. Thus, we propose that Cyclin F helps to limit centrosomeduplication to once per cell cycle to maintain chromosome stability.

Cyclin E is necessary for centrosome duplication (by an unknownmechanism)21,22. Our results indicate that Cyclin F limits centrosomeduplication by targeting CP110 for proteolysis. Thus, Cyclin E andCyclin F have opposing roles, explaining why the former is expressedin S phase (when centrosome duplication occurs) and the latter in G2phase (when centrosome duplication is restricted).

In summary, our study shows that Cyclin F forms an active SCFubiquitin ligase complex (through its F-box motif), and it binds

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Figure 3 | Cyclin F silencing induces centrosome and mitotic aberrations.a, U-2OS cells were transfected with siRNAs to a LacZ, Cyclin F, or bothCyclin F and CP110, synchronized as in Fig. 1a, fixed at the indicated timesafter release from the block, and incubated with anti-Centrin 2 antibody(red) and anti-c-tubulin antibody (green). DNA was stained with DAPI.Insets show magnified views of centrosomes. Scale bar, 10 mm. The graphs onthe right show quantification of three experiments. Error bars indicate 6s.d.

*P 5 0.003; **P 5 0.001; ***P , 0.001. b, Experiments were performed asin a, except that cells were collected 14 h after release from the block andstained with anti-Centrin 2 antibody (red) and anti-a-tubulin antibody(green). Scale bar, 5 mm. The graphs on the right show the percentages ofcells with various abnormal mitotic phenotypes. Error bars indicate 6s.d.*P , 0.001.

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CP110 using a mode of substrate recognition identical to that used bythe canonical cyclins involved in protein phosphorylation (that is,through the hydrophobic patch in the cyclin box domain and the RxLmotif in the substrate), thereby unifying the functions of the twoCyclin F homology domains. We show both temporal and spatialregulation of the SCFCyclin F-mediated proteolysis of CP110 and dem-onstrate that this event has a physiological role in controlling genomeintegrity.

METHODS SUMMARYBiochemical methods. Extract preparation, immunoprecipitation, and immu-

noblotting have been described23,24.

Plasmids. CP110 cDNA was from OpenBioSystems (clone MHS1010-7295919).

Cyclin F cDNA was amplified by PCR using a cDNA library from HEK-293T

cells. CP110 and Cyclin F mutants were generated either using the QuikChange

Site-directed Mutagenesis kit (Stratagene) or by standard PCR methods. pCBF

GFP-CP110 WT and CP110 truncation mutants were as described10,13.

Immunofluorescence microscopy. For indirect immunofluorescence staining,

cells were grown on glass coverslips and then fixed with methanol for 10 min. Thecells were permeabilized with PBS/1% Triton X-100 for 10 min and blocked for

1 h in PBS/0.1% Triton X-100 containing 3% BSA before incubation with prim-

ary antibodies. Alexa Fluor 568-conjugated goat anti-rabbit and Alexa Fluor 488-

conjugated goat anti-mouse IgGs (Invitrogen) were used as secondary antibod-

ies. DAPI was used to counterstain DNA. Slides were mounted with Prolong-

Gold (Invitrogen). Image acquisition was performed using a Zeiss Axiovert

200 M microscope (63 3 objective lens, N.A. 1.4, 1.6 3 Optovar), equipped with

a cooled Retiga 2000R CCD (QImaging). Mitotic images were deconvoluted

using Metamorph (Molecular Devices). The images represent the maximum

projection of 12 deconvoluted planes. Confocal microscopy (used in Fig. 2

and Supplementary Fig. 4) was performed using a Zeiss LSM 510, equipped with

Zeiss LSM 510 software.

Full Methods and any associated references are available in the online version ofthe paper at www.nature.com/nature.

Received 18 January; accepted 30 April 2010.

1. Cardozo, T. & Pagano, M. The SCF ubiquitin ligase: insights into a molecularmachine. Nature Rev. Mol. Cell Biol. 5, 739–751 (2004).

2. Petroski, M. D. & Deshaies, R. J. Function and regulation of cullin-RING ubiquitinligases. Nature Rev. Mol. Cell Biol. 6, 9–20 (2005).

3. Jin, J. et al. Systematic analysis and nomenclature of mammalian F-box proteins.Genes Dev. 18, 2573–2580 (2004).

4. Skaar, J. R., D’Angiolella, V., Pagan, J. K. & Pagano, M. SnapShot: F box proteins II.Cell 137, 1358–1358 (2009).

5. Bai, C. et al. Skp1 connects cell cycle regulators to the ubiquitin proteolysismachinery through a novel motif, the F-box. Cell 86, 263–274 (1996).

6. Fung, T. K., Siu, W. Y., Yam, C. H., Lau, A. & Poon, R. Y. Cyclin F is degraded duringG2-M by mechanisms fundamentally different from other cyclins. J. Biol. Chem.277, 35140–35149 (2002).

7. Tetzlaff, M. T. et al. Cyclin F disruption compromises placental development andaffects normal cell cycle execution. Mol. Cell. Biol. 24, 2487–2498 (2004).

8. Florens, L. & Washburn, M. P. Proteomic analysis by multidimensional proteinidentification technology. Methods Mol. Biol. 328, 159–175 (2006).

9. Bai, C., Richman, R. & Elledge, S. J. Human cyclin F. EMBO J. 13, 6087–6098(1994).

10. Chen, Z., Indjeian, V. B., McManus, M., Wang, L. & Dynlacht, B. D. CP110, a cellcycle-dependent CDK substrate, regulates centrosome duplication in humancells. Dev. Cell 3, 339–350 (2002).

11. Kleylein-Sohn, J. et al. Plk4-induced centriole biogenesis in human cells. Dev. Cell13, 190–202 (2007).

12. Dobbelaere, J. et al. A genome-wide RNAi screen to dissect centriole duplicationand centrosome maturation in Drosophila. PLoS Biol. 6, e224 (2008).

13. Spektor, A., Tsang, W. Y., Khoo, D. & Dynlacht, B. D. Cep97 and CP110 suppress acilia assembly program. Cell 130, 678–690 (2007).

14. Kohlmaier, G. et al. Overly long centrioles and defective cell division upon excessof the SAS-4-related protein CPAP. Curr. Biol. 19, 1012–1018 (2009).

*

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Figure 4 | The failure to degrade CP110 causes centrosome and mitoticdefects. a, HeLa cells were transfected with FLAG–CP110 orFLAG–CP110(RxL/AxA), synchronized at G1/S as in Fig. 1a, collected at theindicated times, lysed, and immunoblotted as indicated. b, U-2OS cells weretransfected with FLAG-CP110 or FLAG-CP110(RxL/AxA), collected 48 hafter transfection, and stained with anti-FLAG antibody to visualize CP110(red) and anti-c-tubulin (green) antibody. Insets show magnified views ofcentrosomes. Scale bar, 10 mm. The graphs on the right show quantificationof cells with excess Centrin 2 and c-tubulin dots. Error bars indicate 6s.d.*P , 0.001 (n 5 3). c, Experiments were performed as in b, except that cellswere transfected with only FLAG–CP110(RxL/AxA), collected 14 h after

release from the block, and stained with anti-a-tubulin antibody (green) andanti-FLAG antibody to visualize CP110-positive cells (red). The yellow inmerged images shows colocalization of CP110 and a-tubulin (yellowarrows). Green arrows show spindles negative for CP110. CP110-negativeand CP110-positive cells from the same coverslip are shown. Scale bar, 5mm.d, U-2OS cells were transfected with an empty vector (EV) or FLAG-CP110(RxL/AxA), fixed, and stained with DAPI to visualize the DNA.Micronuclei are highlighted by arrows. The graph on the bottom showsquantification of cells containing micronuclei. Error bars indicate 6s.d.*P , 0.0001.

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15. Schulman, B. A., Lindstrom, D. L. & Harlow, E. Substrate recruitment to cyclin-dependent kinase 2 by a multipurpose docking site on cyclin A. Proc. Natl Acad. Sci.USA 95, 10453–10458 (1998).

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17. Quintyne, N. J., Reing, J. E., Hoffelder, D. R., Gollin, S. M. & Saunders, W. S. Spindlemultipolarity is prevented by centrosomal clustering. Science 307, 127–129 (2005).

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Supplementary Information is linked to the online version of the paper atwww.nature.com/nature.

Acknowledgements We thank S. Elledge for Cyclin FFlox/– and Cyclin F2/2 MEFs,J.R. Skaar for reading the manuscript and F.M. Forrester for technical help. M.P. isgrateful to T.M. Thor for continuous support. This work was funded by fellowshipsfrom the American Italian Cancer Foundation to V.D’A. and V.D., a grant from theMarch of Dimes (1-FY08-372) to B.D. and grants from the National Institutes ofHealth (R01-GM057587, R37-CA076584 and R21-AG032560) to M.P. A.S., L.F.and M.P.W. are supported by the Stowers Institute for Medical Research. V.D’A. isa Leukemia & Lymphoma Society Fellow. M.P. is an Investigator with the HowardHughes Medical Institute.

Author Contributions V.D’A. and V.D. performed and planned all experiments andhelped to write the manuscript. M.P. coordinated the study, oversaw the results,and wrote the manuscript. S.V. and B.D. provided reagents, advice and assistancewith the analysis of c-tubulin and Centrin 2 foci. A.S., L.F. and M.P.W. performedthe mass spectrometry analysis of the Cyclin F complex purified by V.D’A. Allauthors discussed the results and commented on the manuscript.

Author Information Reprints and permissions information is available atwww.nature.com/reprints. The authors declare no competing financial interests.Readers are welcome to comment on the online version of this article atwww.nature.com/nature Correspondence and requests for materials should beaddressed to M.P. ([email protected]).

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mailto:[email protected]

METHODSCell culture and cell cycle synchronization. HeLa, U-2OS, RPE1-hTERT, HEK-

293T and H1299, HCT116 cells were maintained in Dulbecco’s modified Eagle’s

medium containing 10% fetal bovine serum (FBS). For synchronization at G1/S,

HeLa cells were cultured in the presence of 2 mM thymidine (Sigma) for 16 h,

washed twice with PBS, and cultured in fresh medium without thymidine for 8 h.

After another 16 h in thymidine, cells were washed twice with PBS and cultured

in fresh medium. siRNA oligos were transfected between the first and second

thymidine block. With the exception of experiments shown in Figs 3 and 4c, to

trap cells in prometaphase, nocodazole (100 ng ml–1) was added 5 h after therelease from the thymidine block. In the experiments shown in Fig. 2c and

Supplementary Fig. 1b, we added 10 mM MG132 for 3–6 h before harvesting

the cells.

Transient transfections. HEK-293T cells were transfected using the calcium

phosphate method, as described25. U-2OS and HEK-293T were transfected using

Exgene (Fermentas) according to the manufacturer’s instruction. siRNA

duplexes were transfected into subconfluent U-2OS or HeLa cells using

HiPerfect reagent (QIAGEN) according to the manufacturer’s instructions.

Combined siRNA and DNA transfection was performed using Lipofectamine

2000 (Invitrogen), according to the manufacturer’s instructions.

Purification and MudPIT analysis. HEK-293T cells were transfected with con-

structs encoding either FLAG-HA-tagged wild-type Cyclin F or FLAG-HA-

tagged Cyclin F(1–270). Forty-eight hours after transfection, cells were collected

and lysed in lysis buffer (LB: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM

EDTA, 50 mM NaF, 0.5% NP40, plus protease and phosphatase inhibitors).

Cyclin F and associated proteins were immunopurified with anti-FLAG M2

agarose beads (Sigma). After washing, proteins were eluted twice by competition

with FLAG peptide (Sigma). The eluate was then subjected to a second immu-nopurification with an anti-HA resin (12CA5 monoclonal antibody crosslinked

to protein G Sepharose), before elution by competition with HA peptide

(Roche). The final eluate was then precipitated with TCA.

TCA-precipitated proteins were urea-denatured, reduced, alkylated and

digested with endoproteinase Lys-C (Roche), followed by modified trypsin

(Roche), as described8,26. Peptide mixtures were loaded onto 100-mm fused silica

microcapillary columns packed with 5-mm C18 reverse phase (Aqua,

Phenomenex), strong cation exchange particles (Partisphere SCX, Whatman),

and reverse phase27. Loaded microcapillary columns were placed in-line with a

Quaternary Agilent 1100 series HPLC pump and a LTQ linear ion trap mass

spectrometer equipped with a nano-LC electrospray ionization source

(ThermoFinnigan). Fully automated 10-step MudPIT runs were carried out on

the electrosprayed peptides, as described8. Tandem mass (MS/MS) spectra were

interpreted using SEQUEST28 against a database of 61,430 sequences, consisting

of 30,552 human proteins (downloaded from NCBI on 3 April 2008, 177 usual

contaminants (such as human keratins, IgGs and proteolytic enzymes), and, to

estimate false discovery rates, 30,712 randomized amino-acid sequences derived

from each non-redundant protein entry. Peptide/spectrum matches were sortedand selected using DTASelect29 with the following criteria set: spectra/peptide

matches were only retained if they had a DeltCn of at least 0.08 and a minimum

XCorr of 1.8 for singly-, 2.0 for doubly-, and 3.0 for triply-charged spectra. In

addition, peptides had to be fully tryptic and at least seven amino acids long.

Combining all runs, proteins had to be detected by at least two such peptides, or

one peptide with two independent spectra. Under these criteria the final FDRs at

the protein and spectral levels were 1.6% and 0.13% 6 0.05, respectively. Peptide

hits from multiple runs were compared using CONTRAST29. To estimate relative

protein levels, Normalized Spectral Abundance Factors (NSAFs) were calculated

for each detected protein, as described30–32.

Antibodies. We used the following rabbit polyclonal antibodies: Cyclin F (C-20;

Santa Cruz Biotechnology), CP110 (A301-343A; Bethyl Laboratories), Skp1 (H-

163; Santa Cruz Biotechnology), Centrin 2(N-17; Santa Cruz Biotechnology),

phosphorylated Cdc2 (phospho-Tyr15; Santa Cruz Biotechnology), phosphory-

lated Histone H3 (phospho-Ser10; Millipore), p27 (Invitrogen), Cul1

(Invitrogen) and FLAG (Sigma). A second rabbit polyclonal antibody to

CP110 and rabbit polyclonal antibodies against Cep97, Cyclin A, Cyclin B,

and the PSTAIRE peptide have been described10,33,34. The following mouse

monoclonal antibodies were used: Cyclin F (clone 2123D1a, ab50811;

Abcam), a-tubulin (T5168; Sigma), and c-tubulin (T5326; Sigma).

Gene silencing by siRNA. The sequences of oligonucleotides 1, 2 and 3, corres-

ponding to the Cyclin F mRNA, were CCAGUUGUGUGCUGCAUUA,

UAGCCUACCUCUACAAUGA and GCACCCGGUUUAUCAGUAA, respect-

ively. The sequence of the CP100 siRNA has been reported13. A dsRNA oligo to

LacZ mRNA (CGUACGCGGAAUACUUCGA) served as a negative control25.

In vitro ubyquitylation assay. FLAG-tagged wild-type Cyclin F or FLAG-tagged

Cyclin F(LP/AA) were transfected into HEK-293T cells. Twenty-four hours after

transfection, cells were incubated with MG132 for 3 h, before lysis. Anti-Flag M2

agarose beads were used to immunoprecipitate the SCFCyclin F complex. The

beads were washed four times in lysis buffer and twice in ubiquitylation reaction

buffer (URB: 10 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2 and 1 mM

DTT). Beads were then used for in vitro ubiquitylation assays, which were per-

formed in a volume of 30ml, containing 2 mM ATP, 5 mM E1 (Boston Biochem),

10 ngml–1 Ubch3, 10 ng ml–1 Ubch5c, 1mM ubiquitin aldehyde and 2.5 mg ml–1

ubiquitin (Sigma). The reactions were incubated at 30 uC for 2 h and analysed by

immunoblotting with antibodies to CP110.

Statistical analyses. All data represent the average from at least three independ-

ent experiments, with at least 100 cells counted per experiment. Significance were

calculated by ANOVA (Figs 3a, 3b and 4b, and Supplementary Figs 7 and 8) or

double-tailed t-test (Fig. 4d, Supplementary Fig. 9) using GraphPad Prism soft-

ware. Differences were considered significant when P was ,0.05.

25. Bashir, T., Dorrello, N. V., Amador, V., Guardavaccaro, D. & Pagano, M. Control ofthe SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase. Nature428, 190–193 (2004).

26. Washburn, M. P., Wolters, D. & Yates, J. R., III. Large-scale analysis of the yeastproteome by multidimensional protein identification technology. NatureBiotechnol. 19, 242–247 (2001).

27. McDonald, W. H. et al. Comparison of three directly coupled HPLC/MS, 2-phaseMud PIT, and 3-phase Mud PIT. Int. J. Mass Spectrom. 219, 245–251 (2002).

28. Eng, J., McCormack, A. L. & Yates, J. R., III. An approach to correlate tandem massspectral data of peptides with amino acid sequences in a protein database.J. Amer. Mass Spectrom. 5, 976–989 (1994).

29. Tabb, D. L., McDonald, W. H. & Yates, J. R., III. DTASelect and Contrast: tools forassembling and comparing protein identifications from shotgun proteomics.J. Proteome Res. 1, 21–26 (2002).

30. Florens, L. et al. Analyzing chromatin remodeling complexes using shotgunproteomics and normalized spectral abundance factors. Methods 40, 303–311(2006).

31. Paoletti, A. C. et al. Quantitative proteomic analysis of distinct mammalianMediator complexes using normalized spectral abundance factors. Proc. NatlAcad. Sci. USA 103, 18928–18933 (2006).

32. Zybailov, B. et al. Statistical analysis of membrane proteome expression changesin Saccharomyces cerevisiae. J. Proteome Res. 5, 2339–2347 (2006).

33. Pagano, M., Pepperkok, R., Verde, F., Ansorge, W. & Draetta, G. Cyclin A isrequired at two points in the human cell cycle. EMBO J. 11, 761–771 (1992).

34. Carrano, A. C. & Pagano, M. Role of the F-box protein Skp2 in adhesion-dependentcell cycle progression. J. Cell Biol. 153, 1381–1389 (2001).

doi:10.1038/nature09140

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www.nature.com/doifinder/10.1038/nature09140



SUPPLEMENTARY INFORMATION

1www.nature.com/nature

doi: 10.1038/nature09140

EV Cycli

n F

Fbxw

1Fb

xw11

Fbxw

4Fb

xw5

Fbxw

7αFb

xw7γ

Fbxw

8

Fbxw

2

CP110

Skp1

FBPs(α-FLAG)

IP: α -FLAG

WCE

EV Cycli

n F

Cycli

n A

CP110

CDKs (α-PSTAIRE)

Cyclin B

p27

cyclins(α-FLAG)

Supplementary Figure 1. D’Angiolella et al.

a

WCE EV Cycli

n F

Fbxo

3Fb

xo7

Fbxo

9Fb

xo11

Fbxo

28Fb

xo46

IP: α-FLAGb

Supplementary Figure 1. Cyclin F interacts with CP110 but not CDKs

a, Cyclin F does not bind CDKs. HEK‐293T cells were transfected with an empty

vector (EV), FLAG‐tagged Cyclin A, or FLAG‐tagged Cylin F. Whole cell extracts

(WCE) were immunoprecipitated (IP) with anti‐FLAG resin, and

immunoprecipitates were probed with antibodies to the indicated proteins. The

anti‐PSTAIRE antibody was generated against the sequence EGVPSTAIREISLLKE, a

16 amino acid sequence present in Cdk1, Cdk2, Cdk3, and other CDKs (ref. 33).

b, Cyclin F specifically interacts with CP110 in cultured cells. HEK‐293T cells

were transfected with empty vector (EV) or the indicated FLAG‐tagged F‐box

protein constructs (FBPs). Whole cell extracts were immunoprecipitated (IP) with

anti‐FLAG resin, and immunoprecipitates were probed with antibodies to the

indicated proteins.


doi: 10.1038/nature09140 SUPPLEMENTARY INFORMATION

Supplementary Figure 1. Cyclin F interacts with CP110 but not CDKs

a, Cyclin F does not bind CDKs. HEK‐293T cells were transfected with an empty

vector (EV), FLAG‐tagged Cyclin A, or FLAG‐tagged Cylin F. Whole cell extracts

(WCE) were immunoprecipitated (IP) with anti‐FLAG resin, and

immunoprecipitates were probed with antibodies to the indicated proteins. The

anti‐PSTAIRE antibody was generated against the sequence EGVPSTAIREISLLKE, a

16 amino acid sequence present in Cdk1, Cdk2, Cdk3, and other CDKs (ref. 33).

b, Cyclin F specifically interacts with CP110 in cultured cells. HEK‐293T cells

were transfected with empty vector (EV) or the indicated FLAG‐tagged F‐box

protein constructs (FBPs). Whole cell extracts were immunoprecipitated (IP) with

anti‐FLAG resin, and immunoprecipitates were probed with antibodies to the

indicated proteins.


SUPPLEMENTARY INFORMATIONdoi: 10.1038/nature09140

IP: α FLAG

WCE

EV CP11

0 W

TCP

110(

1-22

9)

CP11

0(20

0-56

5)

CP11

0(36

5-99

1)

CP11

0(62

0-99

1)

CP11

0(1-

565)

CP11

0(RxL

/AxA

)

CP110(α-FLAG)

RXL

RXL1 991

1 229

RXL

200 565

RXL 991365

991620

RXL1 565

AXA1 991RXL

CP110:

+

-

-

+

-

-

-


Cyclin F

a

bBinding to Cyclin F

Supplementary Figure 2. The RXL cyclin binding motif of CP110 is necessary

for Cyclin F binding

a, HEK‐293T cells were transfected with an empty vector (EV), FLAG‐tagged

wild type CP110, or the indicated FLAG‐tagged CP110 mutants. Whole cell extracts

(WCE) were immunoprecipitated (IP) with anti‐FLAG resin, and immunocomplexes

were probed with antibodies to the indicated proteins.

b, Schematic representation of CP110 mutants. CP110 mutants that interacted

with endogenous Cyclin F are designated with the symbol (+).



55

Cyclin A2: SMRAILVDWLVEV / /LRGKLQLVG

Cyclin B : NMRAILIDWLVQV / /PKKMLQLVG

Cyclin D1: SMRKIVATWMLEV / /KKSRLQLLG

Cyclin E : KMRAILLDWLMEV / /VKTLLQLIG

Cyclin F : TMRYILIDWLVEV / / PRYRLQLLG

209 221 248 256

308 320 348 356

143 155 184 192

67 95 103

200 212 240 248

WCE

EV Cyclin

F W

TCyc

lin F

(1-2

70)

Cyclin

F(1

-549

)

Cyclin

F(1

-600

)

Cyclin

F(1

-650

)

Cyclin

F(1

-750

)

CP110

Skp1

Cyclin F(α-FLAG)


IP:α-FLAG

WCE

Cyclin

F W

TCyc

lin F

(LP/

AA)

EVCP110

Cyclin F(α-FLAG)

Skp1

Cul1

IP:α-FLAG

a

b

1 270

Cyclin F:

1

1

1

1

1 786

1 786

549

600

650

750

+

-

+

+

+

F-box Cyclin domain

LP/AA++

c

Nuclearlocalization

+

1 786(M/A;L/A)

-

+ -

+

+

Binding to CP110

-

Centrosomal localization

270 786

-

+

+

+

+

+

+

+

+

-

+

+

+

+

IP:α-FLAG

Cyclin

F W

TCyc

lin F

(M/A

;L/A)

CP110

Cyclin F(α-FLAG)

Skp1

WCE

d

Supplementary Figure 3. Cyclin F binding to CP110 requires the hydrophobic

patch of Cyclin F

a, Schematic representation of Cyclin F mutants. Binding to endogenous CP110,

centrosomal localization, and nuclear localization of wild type Cyclin F and Cyclin F

mutants are designated with the symbol (+).

b, HEK‐293T cells were transfected with an empty vector (EV) or the indicated

FLAG‐tagged Cyclin F mutants. Whole cell extracts (WCE) were

immunoprecipitated (IP) with anti‐FLAG resin, and immunocomplexes were probed

with antibodies to the indicated proteins.

c,. Alignment of the amino acid regions corresponding to the hydrophobic patch

in human Cyclin F and the previously reported hydrophobic patches (which are

responsible for the binding to the RXL motif of substrates) in other cyclins

(highlighted in yellow).

d, HEK‐293T cells were transfected with either wild type, FLAG‐tagged Cyclin F, or

FLAG‐tagged Cyclin F(M/A;L/A), a mutant in the hydrophobic patch. Whole cell

extracts (WCE) were immunoprecipitated (IP) with anti‐FLAG resin, and

immunocomplexes were probed with antibodies to the indicated proteins.



Supplementary Figure 3. Cyclin F binding to CP110 requires the hydrophobic

patch of Cyclin F

a, Schematic representation of Cyclin F mutants. Binding to endogenous CP110,

centrosomal localization, and nuclear localization of wild type Cyclin F and Cyclin F

mutants are designated with the symbol (+).

b, HEK‐293T cells were transfected with an empty vector (EV) or the indicated

FLAG‐tagged Cyclin F mutants. Whole cell extracts (WCE) were

immunoprecipitated (IP) with anti‐FLAG resin, and immunocomplexes were probed


c,. Alignment of the amino acid regions corresponding to the hydrophobic patch

in human Cyclin F and the previously reported hydrophobic patches (which are

responsible for the binding to the RXL motif of substrates) in other cyclins

(highlighted in yellow).

d, HEK‐293T cells were transfected with either wild type, FLAG‐tagged Cyclin F, or

FLAG‐tagged Cyclin F(M/A;L/A), a mutant in the hydrophobic patch. Whole cell

extracts (WCE) were immunoprecipitated (IP) with anti‐FLAG resin, and

immunocomplexes were probed with antibodies to the indicated proteins.



S phase G2 phase


Supplementary Figure 4. CP110 levels on the centrioles decrease in G2 cells

U‐2OS cells were synchronized at G1/S using a double‐thymidine block before

release into fresh medium. Cells were then fixed and incubated with an anti‐CP110

antibody (red) and anti‐γ‐tubulin antibody (green). DNA was stained with DAPI.

Insets show magnified views of the two centrosomes (arrows). Scale bar = 10 µM.

Confocal microscopy was used to visualize the cells. The figure shows

representative cells in S and G2. In S phase, the two γ‐tubulin dots are distinct but

adjacent. In G2, they are clearly separated by at least 2 µM.



Cyclin

F

EV Cyclin

F

EV Cyclin

F

EV

CP110

Skp1

Cyclin F(α-FLAG)


HCT116 HeLa H1299

EV

Cyclin

F

Cyclin

F(M

/A;L/

A)U-2OS

Supplementary Figure 5. Forced expression of Cyclin F induces a reduction in

CP110 levels

U‐2OS, HeLa, HCT116, and H1299 cells were transfected with an empty vector (EV)

or constructs encoding FLAG‐tagged, wild type Cyclin F, or FLAG‐tagged Cyclin

F(M/A;L/A), as indicated. Forty‐eight hours after transfection, cells were collected,

lysed, and immunoblotted with antibodies to the indicated proteins.




siCyclin F(oligo#2)

0 5 7 9 12 14

siLacZ

0 5 7 9 12 14 Hrs

CP110

Cyclin F

Cyclin A

Skp1

CEP97

pCdc2 (Y15)

siCyclin F(oligo#1)

0 5 7 9 12 14

siLacZ

0 5 7 9 12 14 Hrs

CP110

Cyclin F

pHH3 (S10)

Cyclin A

Skp1

siCyclin F(oligo#3)

0 5 7 9 12 14

a

b

siCyclin FsiLacZ

Hrs8 16 24 32 34 360 24 32 34 36

CP110

Cep97

Cyclin F

Cyclin B1

pHH3(S10)

pCdc2(Y15)

2n97.1 95.1 18.2 12.3 6.8 6.5 6.5 13.4 6.4 9.38 7.42

0.8 2.1 63.7 30.2 14.8 10.8 4.8 28.2 11.1 9.45 4.05

2.1 2.8 18.1 57.5 78.4 82.7 88.7 58.4 82.5 81.1 88.5

Skp1

c

>2n<4n

4n

Supplementary Figure 6. Silencing of Cyclin F in G2 results in CP110

stabilization

a, U‐2OS cells were transfected with short interfering RNAs (siRNAs) to either a

non‐relevant mRNA (LacZ) or Cyclin F mRNA (oligo #2). Cells were then

synchronized at G1/S using a double‐thymidine block before release into fresh

medium. Cells were then collected at the indicated times, lysed, and immunoblotted


b, HeLa cells were transfected with siRNAs to either a non‐relevant mRNA

(LacZ) or Cyclin F mRNA (oligos # 1 and 3, as indicated). Cells were then

synchronized at G1/S using a double‐thymidine block before release into fresh

medium. Cells were then collected at the indicated times, lysed, and immunoblotted


c, RPE1‐hTERT cells were transfected with siRNAs to either a non‐relevant mRNA

(LacZ) or Cyclin F mRNA (oligo #2). Cells were then synchronized in G0/G1 by

serum starvation for 72 hours before release into fresh medium containing serum.

Cells were then collected at the indicated times, lysed, and immunoblotted with

antibodies to the indicated proteins. DNA content was monitored by flow

cytometry.



EV EV Cyclin

F

C

yclin

F +

CP110(R

XL/AXA)

Hydroxyurea-48 Hrs

0

5

10

15

20

25

Centrin 2

γ-tubulin

EV EV Cyclin F Cyclin F+CP110(RXL/AXA)

CP110

Cyclin F

pChk1(S317)

Skp1

+ HU 48 Hrs

Per

cent

age

of c

ells

with

>2 γ-

tubu

lin d

ots

o

r >4

Cen

trin2

dot

s


*

**

Supplementary Figure 7. Cyclin F overexpression inhibits centrosome

reduplication in S phasearrested cells

U‐2OS cells were transfected with an empty vector (EV) or a construct encoding

FLAG‐tagged Cyclin F in the presence or absence of CP110(RxL/AxA), a mutant that

does not interact with Cyclin F. Cells were then treated for 48 hours with

hydroxyurea, to induce a block in S phase, before harvesting, lysis, and

immunoblotting with antibodies to the indicated proteins. The graph shows the

percentages of cells with excess Centrin 2 foci (more than four per cell) and excess

γ‐tubulin dots (more than two per cell). The data represent the average from three

independent experiments, with at least 100 cells counted per experiment. Error

bars indicate +/‐SD. *= p=0.002; **= p=0.001 (calculated by ANOVA).




CP110 γ-tubulin

siLacZ siCyclin F

merge CP110 γ-tubulin merge

Per

cent

age

of c

ells

with

>4

CP

110

dots

siLacZ siCyclinF

+Cyclin F WT +Cyclin F(M/A;L/A)0

5

10

15

20

25

30

35

*

Supplementary Figure 8. Silencing of Cyclin F induces an increase in the

number of CP110 foci

U‐2OS cells were transfected with siRNAs to a non‐relevant mRNA (LacZ) or to

Cyclin F mRNA and synchronized by a double‐thymidine block. Cells were fixed at

nine hours after release from the block and incubated with an anti‐CP110 antibody

(red) and an anti‐γ‐tubulin antibody (green). DNA was stained with DAPI. Insets

show magnified views of centrosomes. Scale bar = 10 µM. In parallel experiments,

cells treated with siRNA oligos to Cyclin F were also transfected with constructs

encoding either a wild type, but siRNA‐insensitive, Cyclin F or an siRNA‐resistant

Cyclin F mutant unable to bind CP110 [Cyclin F(M/A;L/A)]. The graph shows the

percentages of cells with excess CP110 dots (more than four per cell). The data

represent the average from three independent experiments, with at least 100 cells

counted per experiment. Error bars indicate +/‐SD. *= p=0.001 (calculated with

ANOVA).



Cyclin F Cyclin F

mergeCentrin2 γ-tubulinmergeCentrin2 γ-tubulin

Centrin2

γ-tubulin

0

2

4

6

8

10

12

14

Flox/- -/-

CP110

Skp1

Per

cent

age

of c

ells

with

>2 γ-

tubu

lin d

ots

a

nd >

4 C

entri

n2 d

ots


Flox/- -/-

Cyclin F Cyclin F Flox/- -/-

*

*

Supplementary Figure 9. Cyclin F null mouse embryonic fibroblasts (MEFs)

display CP110 accumulation and increased centrosome number

Cyclin F‐/‐ and parental Cyclin FFlox/‐ MEFs were fixed and incubated with an anti‐

Centrin 2 antibody (red) and an anti‐γ‐tubulin antibody (green). DNA was stained

with DAPI. Insets show magnified views of centrosomes. Scale bar = 10 µM. The

graph shows the percentages of cells with excess Centrin 2 foci (more than four per

cell) and excess γ‐tubulin dots (more than two per cell). The data represent the

average from three independent experiments, with at least 100 cells counted per

experiment. Error bars indicate +/‐SD. *= p<0.0001 (calculated by double tailed t‐

test). Bottom panels show Cyclin F‐/‐ and parental Cyclin FFlox/‐ MEFs that were

lysed and immunoblotted with antibodies to the indicated proteins.




siLacZ siCyclin F

0102030405060708090

100

Bipolar

Monop

olar

Multipo

lar

Asymmetri

c bipolar

lagging chro

mosomes

siLacZ

siCyclin F

siCyclin F + siCP110

**

**

*

Cel

ls (p

erce

ntag

e)

Supplementary Figure 10. Silencing of Cyclin F induces mitotic aberrations

HeLa cells were transfected with siRNAs to a non‐relevant mRNA (LacZ) or Cyclin F

mRNA and synchronized by a double‐thymidine block. Cells were fixed nine hours

after release from the block and stained with an anti‐Centrin 2 antibody (red) and

an anti‐α‐tubulin antibody (green). DNA was stained with DAPI. The yellow color

in merged images shows colocalization of Centrin 2 and α‐tubulin. Scale bar = 5 µM.

The graph shows the percentages of cells with various abnormal mitotic

phenotypes. The data represent the average from three independent experiments,

with at least 100 cells counted per experiment. Error bars indicate +/‐SD. *=

p<0.003; **= p=0.001 (calculated by ANOVA)



HeLaU-2OS

siLac

Z

siCyc

lin F

siC

yclin

F+s

iCP11

0

siLac

ZsiC

yclin

F

siCyc

lin F

+siC

P110

CP110

Cyclin F

Skp1

Supplemntary Figure 11. D’Angiolella et al.

Supplementary Figure 11. Cyclin F knockdown results in increased levels of

CP110 in asynchronous cells

U‐2OS (left panels) and HeLa cells (right panels) were transfected with siRNAs to

either a non‐relevant mRNA (LacZ), Cyclin F mRNA, or both. Cells were collected

after 48 hours, lysed, and immunoblotted with antibodies to the indicated proteins.



Supplementary Table 1. MudPIT analysis of two Cyclin F immunopurifications,

listing normalized spectral abundance factors (NSAFs; ref. 32) for the indicated

proteins.

Cyclin F WT Cyclin F(1-270) Control IP

Protein

dNSAF

AVG

Detected

# Out of

2

dNSAF

AVG

Detected

# Out of

2

dNSAF

AVG

Detected

# Out of

4

Cyclin F 0.127895 2 0.040986 2 0 0

Skp1 0.258895 2 0.120016 2 0.000714 1

Cul1 0.010134 2 0.000216 1 0 0

CP110 0.00037 2 0 0 0 0

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