HOPS is an essential constituent of centrosome assembly

Cell Cycle ISSN: 1538-4101 (Print) 1551-4005 (Online) Journal homepage: https://www.tandfonline.com/loi/kccy20 HOPS is an essential constituent of centrosome assembly Stefania Pieroni, Maria Agnese Della Fazia, Marilena Castelli, Danilo Piobbico, Daniela Bartoli, Cinzia Brunacci, Marina Maria Bellet, Mariapia Viola-Magni & Giuseppe Servillo To cite this article: Stefania Pieroni, Maria Agnese Della Fazia, Marilena Castelli, Danilo Piobbico, Daniela Bartoli, Cinzia Brunacci, Marina Maria Bellet, Mariapia Viola-Magni & Giuseppe Servillo (2008) HOPS is an essential constituent of centrosome assembly, Cell Cycle, 7:10, 1462-1466, DOI: 10.4161/cc.7.10.5882 To link to this article: https://doi.org/10.4161/cc.7.10.5882 Published online: 03 Jun 2008. Submit your article to this journal Article views: 143 View related articles Citing articles: 12 View citing articles Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=kccy20 [Cell Cycle 7:10, 1462-1466; 15 May 2008]; ©2008 Landes Bioscience Report HOPS is an essential constituent of centrosome assembly Stefania Pieroni,1,† Maria Agnese Della Fazia,1,† Marilena Castelli,1 Danilo Piobbico,1 Daniela Bartoli,1 Cinzia Brunacci1, Marina Maria Bellet,1 Mariapia Viola-Magni1 and Giuseppe Servillo1,* Università degli Studi di Perugia; Dipartimento di Medicina Clinica e Sperimentale; Policlinico Monteluce; CEMIN; Perugia Italy †These authors have contributed equally to this work. .D ON OT DI ST Remarkably, proteins involved in the nucleus-cytoplasmic transport play a pivotal role in spindle assembly.8,9 CRM-1 is one of the most important exportin that, through its binding to the nuclear export sites (NES) on cargo proteins, allows the export of specific proteins to the cytoplasm.10 Localization of CRM-1 and Ran in kinetochores and centrosomes11,12 as its role in mitotic spindle assembly and centrosome duplication13,14 underlines the importance of the association between nucleus-cytoplasmic shuttling and mitosis. Moreover, several proteins shuttled by CRM-1 from the nucleus to the cytoplasm are associated with the centrosome.11 HOPS (Hepatocyte Odd Protein Shuttling) is a recently identified protein containing a putative NES and an ubiquitin domain.15 HOPS is overexpressed in residual hepatocytes following partial hepatectomy and it changes localization in relation to the proliferative state of the cell. HOPS shuttles from nucleus to cytoplasm via CRM-1. Overexpression of HOPS induces arrest of proliferation.15 Following the demonstration of HOPS binding to γ-tubulin and CRM-1, we aimed at unravelling the role of HOPS in centrosome function. For this, we knocked down HOPS expression by small interfering RNA (siRNA) in cells. Strikingly, our results show that inhibition of HOPS expression leads to: (i) abnormal centrosome amplification; (ii) multinucleated cells accumulation and (iii) abnormal spindle formation. Furthermore, we provided evidence that HOPS is associated to CRM-1, γ-tubulin, eEF-1A and HSP70 in both the centrosome and cytosolic complexes. Previous investigations suggested that centrosomal proteins are not assembled de novo in centrosomes, but they are associated in cytosolic complexes that can be rapidly recruited for centrosome duplication.16 Our results assign to HOPS an important role in centrosome duplication and maintenance of genomic stability. BIO SC IEN CE Centrosomes direct microtubule organization during cell division. Aberrant number of centrosomes results from alteration of its components and leads to abnormal mitoses and chromosome instability. HOPS is a newly discovered protein isolated during liver regeneration, implicated in cell proliferation. Here, we provide evidence that HOPS is an integral constituent of centrosomes. HOPS is associated with classical markers of centrosomes and found in cytosolic complexes containing CRM-1, γ-tubulin, eEF-1A and HSP70. These features suggest that HOPS is involved in centrosome assembly and maintenance. HOPS depletion generates supernumerary centrosomes, multinucleated cells and multipolar spindle formation leading to activation of p53 checkpoint and cell cycle arrest. The presence of HOPS in cytosolic complexes supports that centrosome proteins might be preassembled in the cytoplasm to then be rapidly recruited for centrosome duplication. Altogether these data show HOPS implication in the control of cell division. HOPS contribution appears relevant to understand genomic instability and centrosome amplification in cancer. RIB UT E . Key words: centrosome, HOPS, cell cycle, CRM-1, mitotic spindle Introduction © 20 08 LA ND ES In animal cell the centrosome is a small organelle formed by two centrioles surrounded by different proteins indicated as pericentriolar material (PCM).1,2 The strictly controlled presence of two centrosomes at the beginning of mitosis guarantees the appropriate formation of the mitotic spindle.3,4 The centrosome has been hypothesized to be an essential guardian of cell cycle progression, although this role has not been clearly defined at the molecular level.2,5 Centrosomes start duplicating in the late G1/S phase, allowing the formation of procentrioles. During the S and G2 phases the procentrioles recruit the PCM proteins until the centrosomes are entirely duplicated. The mechanism, by which the centrosome is rapidly duplicated and activated during cell cycle, is presently unclear. Perturbations in centrosome duplication lead to abnormal amplification of centrosomes, multipolar spindles and genomic instability.6,7 *Correspondence to: Giuseppe Servillo; Dipartimento di Medicina Clinica e Sperimentale; Policlinico Monteluce; Perugia 06122 Italy; Tel.: 39.075.5720655; Fax: 39.075.5726803; Email: [email protected] Submitted: 02/21/08; Accepted: 03/07/08 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/5882 1462 Results and Discussion HOPS in cell cycle. HOPS has been described as a protein involved in liver regeneration.15 To analyze HOPS function during cell proliferation, we studied HOPS localization and expression throughout the cell cycle. Immunohistochemical analyses showed that HOPS colocalizes with γ-tubulin, in H35 and NIH-3T3 cells, specifically during the G0/G1 phase, but not during the S and G2/M phases (Fig. 1A; Suppl. Fig. 1 online). Western blot analyses showed a reduction of 50% (densitometric analysis, data not shown) of HOPS expression in S-phase with respect to G2/M phase in cells arrested by aphidicolin and nocodazole treatments, respectively Cell Cycle 2008; Vol. 7 Issue 10 Running title © 20 08 LA ND ES BIO SC IEN CE .D ON OT DI ST RIB UT E . (Fig. 1B). This is in support of the rapid decrease of HOPS expression observed during the S-phase of cells blocked in G0/G1 and released from the arrest.15 These data suggest a dynamic participation of HOPS in the cell cycle. HOPS is localized in centrosomes. Next, we performed immunoprecipitation assays using H35 cell extracts to investigate HOPS interaction with γ-tubulin. Interestingly, HOPS antibody were able to immunoprecipitate γ-tubulin from total cell lysates (Fig. 1C), indicating a specific interaction between these two proteins. Centrosomes were then isolated by a discontinuous sucrose gradient from extracts of H35 cells treated with aphidicholin/nocodazole.17 Fractions of centrosome preparations analyzed by western blot revealed cosedimentation of HOPS, γ-tubulin and CRM-1 in the same fractions (Fig. 1D). The association of centrosome proteins with HOPS, in cells treated with nocodazole, strongly suggests that HOPS is a centrosomal protein. HOPS depletion leads to an abnormal centrosome amplification. To assess the role played by HOPS in the centrosome function, we performed siRNA experiments in NIH-3T3 cells. Hops-siRNA treated cells were analyzed at 24, 48 and 96 hours after transfection. HOPS-depleted cells were compared to untreated cells and scramble-siRNA transfected cells. HOPS siRNA reduced HOPS expression by 70% while Figure 1. HOPS localization throughout cell cycle and in centrosomes. (A) H35 control cells did not show any reduction (Fig. 2A and B). cells were stained with HOPS and γ-tubulin antibodies in different phases of cell Following HOPS silencing, we evaluated whether this cycle. Bar is 10 μm. (B) Western blot analysis of H35 cells arrested in S and G /M 2 perturbation might interfere with centrosome formation. phases by aphidicholin/nocodazole treatments. CyclinA and Ser10 phosphorylated To do this, we counted the number of centrosomes per cell H3 histone (H3S10ph) antibodies were used as S phase and G2/M phase control using γ-tubulin (Fig. 2C). In addition, to assess a centrosome respectively. β-tubulin antibody was used as loading control. c: untreated cells. (C) hyperamplification, we used two other centrosome markers, HOPS coimmunoprecipitation with γ-tubulin antibody in H35 cells. Total lysate was centrin and pericentrin (Fig. 2D; Suppl. Fig. 2 online). immunoprecipitated with HOPS antibody (IP) and preimmune serum (PS). Resulting immunocomplexes were tested by immunoblotting using γ-tubulin antibody. c: total Centrosome’s amplification gives rise to cytokinesis failure, protein extracts. (D) Centrosome isolation from H35 cells. Immunoblot analysis on errors in chromosomes segregation and aberrant mitosis.6,7 gradient resulting fractions using HOPS, γ-tubulin, CRM-1 antibodies. Lamin B recepInterestingly, while 24 hours after HOPS depletion no effects tor (LBR) and H3S10ph antibodies were used as centrosome purification controls. c: on centrosomes were observed, 48 and 96 hours later we total protein extracts. found a centrosome amplification as demonstrated by the number of cells with more than two centrosomes which rose to 23% and scramble-siRNA transfected cells were estimated at 1.3% at 48 and 21%, respectively. Untreated and scramble-siRNA transfected and 96 hours (Fig. 3C). Remarkably, the increase of multipolar cells displayed the same number of centrosomes as at 24 hours (10% spindles in HOPS depleted cells was independent from the number and 12%) (Fig. 2C). of mitotic figures. These results show that HOPS depletion leads to We next analyzed the presence of multinucleated cells and a striking arrest of cell proliferation. multipolar spindles formation. 96 hours after HOPS silencing, Importantly, HOPS depleted cells arrested their proliferation in multinucleated cells represented 7% with respect to 0.9% in control the G0/G1 phase (62.7%) with a substantial reduction of cells in cells. Immunofluorescence analyses showed that Hops-siRNA treated G2/M phase (4.9%) 96 hours after silencing (Fig. 3D). Recently, it cells exhibited abnormal cell division, resulting in the formation of has been described that the lack of proteins involved in centrosome binucleated or multinucleated cells (Fig. 2E and F). assembling activates p53-dependent cell cycle checkpoint, arresting Daughter cells derived from cells with abnormal spindle are cells in G1 phase.20,21 To verify whether silencing of HOPS leads to aneuploid and present genomic instability.7,18,19 Thus we analyzed activation of the cell cycle checkpoint, we analyzed the expression of spindle alteration and cell division arrest in Hops-siRNA transfected p53 and p21 in Hops-siRNA treated cells. Overexpression of p53 was cells versus untreated cells (Fig. 3A). Notably, the analysis of HOPS- observed 24 hours after HOPS depletion and progressively decreased silenced cells exhibited a significant increase of multipolar spindle afterwards at 48 and 96 hours. p53 activation in turn induced number with respect to the control. In untreated cells multipolar p21, which became overexpressed at 48 and 96 hours after HOPS spindles represent 4.5%, while in Hops-siRNA treated cells this silencing (Fig. 3E). number was dramatically increased from 8.6% at 24 hours to 23,7% Taken together, these observations reveal that HOPS depleat 48 hours, than it decreased to 6% at 96 hours (Fig. 3B). tion leads to defects in centrosome assembly, with an increase of In these cells the number of mitosis counted at 48 and 96 hours centrosomes’ number, formation of multinucleated cells and multiwas 0.6% and 0.02%, respectively. Mitotic figures in untreated cells polar spindles. These alterations very likely might lead to errors during www.landesbioscience.com Cell Cycle 1463 HOPS function in centrosome assembly RIB UT E . Figure 2. HOPS depletion in NIH-3T3 cells. (A) Cells were transfected with Hops-siRNA and scramble-siRNA and analyzed after 24, 48 and 96 hours from depletion. Western blotting was performed using HOPS antibody. β-tubulin antibody was used as loading control. (B) QPCR on Hops-siRNA interferred NIH-3T3 cells. Bars indicate relative quantity of mRNA referred to control sample. A.U. represents arbitrary unit expression referred to control. (C) Diagram in bars showing percentage of cells containing more than two centrosomes. (D) Immunofluorescence staining in HOPS depleted cells with γ-tubulin and centrin antibodies. A larger inset of centrosome is shown in box. Bar is 10 μm. (E) Double immunofluorescence staining in HOPS depleted multinucleated cells using γ-tubulin antibody and DAPI for nuclear staining. Bar is 10 μm. (F) Diagram in bars showing percentage of multinucleated cells. In diagrams: white bars indicate untreated cells, grey bars indicate scramble-siRNA transfected cells, black bars indicate Hops-siRNA depleted cells. © 20 08 LA ND ES BIO SC IEN CE .D ON OT DI ST cell division, resulting in aberrant genome segregation that in turn drives the cell towards mitotic arrest. Similar results have been obtained using a different oligoduplex for HOPS siRNA (data not shown). HOPS in the cytosolic complex. The description of a cytosolic complex as putative precursor of centrosome in which γ-tubulin is associated with eEF-1A and HSP70 in vitro16 and the interaction of HOPS with eEF-1A in vivo15 prompted us to examine the possibility that HOPS might be a component of this complex. Cytosolic extracts from hepatoma cells fractionated by a 40–10% continuous sucrose gradient, revealed a cosedimentation of HOPS with γ-tubulin, eEF-1A and HSP70 in several fractions (Fig. 4A, fractions 6 to 9). The previously observed association of HOPS with CRM-1 and γ-tubulin in centrosomes led us to investigate the presence of CRM-1 with HOPS also in cytosolic complexes. Interestingly, we revealed CRM-1 in the same fractions also containing HOPS, γ-tubulin, eEF-1A and HSP70 (Fig. 4A). Furthermore, to better characterize binding between HOPS and CRM-1, we performed coimmunoprecipitation of HOPS with CRM-1 in the cytosolic complex (Fig. 4B). Our results show that HOPS binds to CRM-1 and together they constitute a complex with γ-tubulin, eEF-1A and HSP70 (Fig. 4B). The identification of cytosolic complexes containing HOPS suggests that essential proteins for centrosome duplication and spindle formation might be assembled in the cytosol where they might become rapidly available for centrosome arrangement and function. A cytoplasmic form Figure 3. HOPS depletion and cell cycle modification. (A) Tri-, tetra- and n-polar spindles images obtained by immunofluorescence staining of Hops-siRNA treated cells using γ-tubulin and α-tubulin of γ-tubulin, associated in a complex, as precursor antibodies. (B) Percentage of abnormal spindles per mitosis at different times following Hops- for centrosome assembly has been described.22-24 siRNA treatment. (C) Percentage of mitotic figures. White and grey bars indicate controls: untreatThis might result into a rapid recruitment of ed cells and cells transfected with scramble-siRNA. Black bars indicate depleted cells transfected using Hops-siRNA. (D) Cell cycle profiles of HOPS depleted NIH-3T3 cells by FACS analysis. Bars γ-tubulin in the centrosome at the onset of indicate the percentage of cells in each mitotic cycle phase. Grey, white and black bars indicate mitosis.25 G0/G1, S and G2/M phases respectively. (E) Western blot analysis on HOPS depleted cells using The organization of a preassembled complex p53 and p21 antibodies. containing γ-tubulin, HOPS, CRM-1, eEF-1A and HSP70 in cytosolic fractions prompted us 1464 Cell Cycle 2008; Vol. 7 Issue 10 © 20 08 LA ND ES BIO SC Cell culture and cell cycle synchronization. H35 hepatoma cells and NIH-3T3 mouse immortalized fibroblasts were maintained and synchronized as previously described.15 Immunofluorescence assays. H35 hepatoma cells, NIH-3T3 and HOPS-depleted-NIH-3T3 cells were fixed and stained using anti-HOPS, anti-γ-tubulin, anti-α-tubulin (Sigma Aldrich), antipericentrin (Santa Cruz Biotechnology), anti-centrin (kind gift of Prof. J.L.Salisbury) antibodies. Antibodies binding was revealed by incubation with appropriate secondary antibodies: Alexa Fluor® 488 FITC-conjugated anti-mouse IgG and Alexa Fluor® 568 Texas-Red® -conjugated anti-rabbit IgG (Molecular Probes™). Nuclei were stained with DAPI (Sigma Aldrich). Images were captured with a Zeiss Axioplan fluorescence microscope controlled by Spot-2 cooled camera (Diagnostic Instruments). Coimmunoprecipitations. H35 hepatoma cells were lysed in RIPA buffer with proteolytic inhibitors (Sigma-Aldrich). 1 mg of whole cell lysate was immunoprecipitated with rabbit polyclonal anti-HOPS antibody as previously described.15 Immunoprecipitation products were probed by immunoblot analysis using mouse monoclonal anti-γ-tubulin antibody. Anti-HOPS reacting cytosolic fractions from sucrose gradients were pooled, dialyzed and subjected to immunoprecipitation with rabbit polyclonal anti-HOPS antibody. Immunoprecipitation products were probed using mouse monoclonal anti-CRM-1 antibody. Centrosome isolation. Centrosomes were isolated from H35 hepatoma cells as described.17 In brief, exponentially growing cells were subjected to cytochalasin D and nocodazole (Sigma-Aldrich) www.landesbioscience.com RIB UT E Figure 4. HOPS in the cytosol and in the centrosome. (A) Western blotting on the cytosolic fractions using HOPS, γ-tubulin, CRM-1, HSP70 and eEF1A antibodies. c: total protein extract. (B) Immunoprecipitation on pooled fractions from 20 to 15% sucrose density (lanes 6–9 Fig. 4A) with HOPS antibody (IP) and preimmune serum (PS). Western blot analysis on immunoprecipitates using anti-CRM-1 antibody. (C) Immunoblot analysis on the centrosome fractions using HOPS, γ-tubulin, CRM-1, HSP70 and eEF-1A antibodies. c: total protein extracts. LBR and H3S10ph antibodies were used as centrosome purification controls. IEN CE Materials and Methods .D ON OT DI ST to investigate whether all of these components are assembled in centrosomes. Western blot analyses performed on centrosome enriched fractions showing cosedimentation of HOPS, CRM-1 and γ-tubulin (Fig. 1A), were found to also contain eEF-1A and HSP70, thus generating a protein macrocomplex of high molecular weight (Fig. 4C, lanes 6 to 11; Suppl. Fig. 3 online). These results support the presence of a previously assembled cytosolic complex that might act as centrosome precursor. The mechanisms by which precursor complexes of centrosomes are recruited during cell duplication have still to be revealed. It has been proposed that cytosolic complexes are targeted to centrosome as precursors,24,25 alternatively the cytosolic complex can regulate centrosome assembly by retaining precursors until centrosome duplication.26 It has been demonstrated that centrosome proteins are present in cytosolic pools and that treatment with cycloheximide does not affect centrosome duplication.27,28 Here, we present the first evidence that HOPS is an essential protein important for centrosome assembly. Knockdown of HOPS leads to hyperamplification of centrosomes, multinucleated cells and multipolar spindles. This in turn results to cell cycle arrest and activation of p53-dependent cell cycle checkpoint. We demonstrate that HOPS is present in cytosolic fractions with well characterized centrosome proteins and that this complex might play a role in centrosome assembly. Future studies will be aimed to identify the mechanisms by which HOPS is recruited and assembled from the cytosol to the centrosome and to elucidate its function during cell division. . HOPS function in centrosome assembly treatment and lysed. Centrosomes were harvested by centrifugation onto a 60% sucrose cushion and centrosomal complexes were further purified by centrifugation through a discontinuous (70%, 50% and 40%) sucrose gradient. Fractions were collected and analyzed. Cytosolic fractions preparation. H35 hepatoma cells were grown to 80% confluence and then arrested in a state resembling late prophase/early metaphase by aphidicolin treatment (Sigma Aldrich) for 16 hours. Cells were washed and incubated for additional 7 hours in fresh medium and then subjected to nocodazole treatment (Sigma Aldrich) for 16 hours. Citosolic complexes were obtained from H-35 cells as previously described16 and loaded onto a continuous sucrose gradients from 40 to 10%. Resulting fractions were collected and analyzed. Immunoblot analysis. H35 hepatoma cells lysate was probed using anti-HOPS antibody. Anti-cyclinA (Santa Cruz Biotechnology) and anti-H3S10ph (Ser10 phosphorylated H3 histone) were used as G2/M and S phase specific markers respectively. HOPS-depleted-NIH-3T3 cells were lysed at different times from transfection and probed using anti-HOPS antibody, anti-p53 (Santa Cruz Biotechnology) and anti-p21 (Dako) antibodies. Antiβ-tubulin (Sigma Aldrich) was used as loading control. Sucrose gradient fractions containing γ-tubulin cytosolic complex and centrosomes were tested by immunoblot analysis using antiHOPS, anti-CRM-1 (Becton Dickinson), anti-γ-tubulin (Sigma Aldrich), anti-eEF1A, anti-HSP70 antibodies (Abcam). All the hybridizations were detected by chemiluminescence ECL™ (Amersham). Cell Cycle 1465 HOPS function in centrosome assembly Acknowledgements . RIB UT E IEN CE We thank E. Borrelli, P.G. Pelicci, S. Brancorsini, E. Ayroldi for the fruitful suggestion and discussion of the paper. We thank J.L. Salisbury for the kind gift of anti-centrin antibody. We also thank S. Pagnotta for the technical assistance. This work was supported by grants from Associazione Italiana Ricerca sul Cancro (AIRC), Fondazione Guido Berlucchi, Ministero della Università e Ricerca (MiUR) prot. 2006061141_003 and Fondazione Cassa di Risparmio di Perugia. 16. Marchesi VT, Ngo N. In vitro assembly of multiprotein complexes containing alpha, beta and gamma tubulin, heat shock protein HSP70, and elongation factor 1 alpha. Proc Natl Acad Sci USA 1993; 90:3028-32. 17. Moudjou M, Bornes M. In Celis JE (ed.), Cell Biology: A laboratory handbook pp 111–119. Academic Press, San Diego, CA 1998. 18. Gisselsson D. Mitotic instability in cancer: is there method in the madness? Cell Cycle 2005; 8:1007-10. 19. D’Assoro AB, Lingue WL, Salisbury JL. Centrosome amplification and the development of cancer. Oncogene 2002; 40:6146-53. 20. Srsen V, Gnadt N, Dammermann A, Merdes A. Inhibition of centrosome protein assembly leads to p53-dependent exit from the cell cycle. J Cell Biol 2006; 174:625-30. 21. Mikule K, Delaval B, Kaldis P, Jurcyzk A, Hergert P, Doxsey S. Loss of centrosome integrity induces p38-p53-p21-dependent G1-S arrest. Nat Cell Biol 2007; 9:160-70. 22. Perret E, Moudjou M, Geraud ML, Derancourt J, Soyer Gobillard MO, Bornens M. Identification of an HSP70-related protein associated with the centrosome from dinoflagellates to human cells. J Cell Sci 1995; 108:711-25. 23. Moudjou M, Bordes N, Paintrand M, Bornens M. gamma-Tubulin in mammalian cells: the centrosomal and the cytosolic forms. J Cell Sci 1996; 109:875-87. 24. Calarco PG. Centrosome precursors in the acentriolar mouse oocyte. Microsc Res Tech 2000; 49:428-34. 25. Khodjakov A, Rieder CL. The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules. J Cell Biol 1999; 146:585-96. 26. Uzawa M, Grams J, Madden B, Toft D, Salisbury JL. Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation. Dev Biol 1995; 171:51-9. 27. Gard DL, Hafezi S, Zhang T, Doxsey SJ. Centrosome duplication continues in cycloheximide-treated Xenopus blastulae in the absence of a detectable cell cycle. J Cell Biol 1990; 110:2033-42. 28. Sluder G, Miller FJ, Cole R, Rieder CL. Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control. J Cell Biol 1990; 110:2025-32. 29. Della Fazia MA, Piobbico D, Bartoli D, Castelli M, Brancorsini S, Viola Magni M, Servillo G. lal-1: a differentially expressed novel gene during proliferation in liver regeneration and in hepatoma cells. Genes to Cells 2002; 7:1183-90. .D ON OT DI ST HOPS depletion by siRNA. Hops-siRNA Stealth™ oligoduplex (target 5'-gctaggagacgacactcagacacta-3') and scramble-siRNA oligoduplex (medium GC content) (Invitrogen) as negative control were transfected into NIH-3T3 cells according to the manufacturer’s instructions. To efficiently deplete HOPS, cells were subjected to a second round of transfection performed 48 hours after the first one. Cells were harvested 24, 48 and 96 hours after the first transfection and processed for further analyses. Data are an average of three separate experiments, shown as mean ± s.d. Quantitative PCR. Total RNA was isolated from controls and HOPS-depleted-NIH-3T3 cells as previously described.29 3 μg of total RNA were retrotranscribed using RevertAid™ H Minus M-MuLV Reverse Transcriptase (Fermentas) and random hexamer primers. Real-Time PCR amplifications were performed using Mx3000P™ Real Time PCR System using Brillant® SYBR® Green QPCR Master Mix (Stratagene) and ROX as reference dye. Hops specific primers were: Forward 5'-TGCTTGCTTGCCTTCTGG-3', Reverse 5'-TGTGCTGGTGTTGTGGTC-3' (Invitrogen). Housekeeping control was mouse HPRT (Hypoxanthine Phosphorybosiltransferase) gene. All the experiments were performed in triplicate. Note BIO SC Supplementary materials can be found at: www.landesbioscience.com/supplement/PieroniCC7-10-Sup.pdf References © 20 08 LA ND ES 1. Doxsey S, McCollum D, Theurkauf W. Centrosomes in cellular regulation. Annu Rev Cell Dev Biol 2005a; 21:411-34. 2. Doxsey S, Zimmerman W, Mikule K. Centrosome control of the cell cycle. Trends Cell Biol 2005b; 6:303-11. 3. Tsou MF, Stearns T. Controlling centrosome number: licenses and blocks. Curr Opin Cell Biol 2006a; 18:74-8. 4. Tsou MF, Stearns T. Mechanism limiting centrosome duplication to once per cell cycle. 2006b; Nature 442:947-51. 5. Krämer A, Lukas J, Bartek J. Checking out the centrosome. Cell Cycle 2004; 11:1390-3. 6. Nigg EA. Centrosome aberrations: cause or consequence of cancer progression? Nat Rev Cancer 2002; 2:815-25. 7. Fukasawa K. Centrosome amplification, chromosome instability and cancer development. Cancer Lett 2005; 230:6-19. 8. Arnaoutov A, Azuma Y, Ribbeck K, Joseph J, Boyarchuk Y, Karpova T, McNally J, Dasso M. Crm1 is a mitotic effector of Ran-GTP in somatic cells. Nat Cell Biol 2005a; 6:626-32. 9. Arnaoutov A and Dasso M. Ran-GTP regulates kinetochore attachment in somatic cells. Cell Cycle 2005b; 9:1161-5. 10. Fornerod M, Ohno M, Yoshida M, Mattaj IW. CRM1 is an export receptor for leucine-rich nuclear export signals. Cell 1997; 90:1051-60. 11. Wang W, Budhu A, Forgues M, Wang XW. Temporal and spatial control of nucleophosmin by the Ran-Crm1 complex in centrosome duplication. Nat Cell Biol 2005; 8:823-30. 12. Weis K. Regulating access to the genome: nucleocytoplasmic transport throughout the cell cycle. Cell 2003; 112:441-51. 13. Di Fiore B, Ciciarello M and Lavia P. Mitotic functions of the Ran GTPase network: the importance of being in the right place at the right time. Cell Cycle 2004; 3:305-13. 14. Budhu AS, Wang XW. Loading and unloading: orchestrating centrosome duplication and spindle assembly by Ran/Crm1. Cell Cycle 2005; 11:1510-4. 15. Della Fazia MA, Castelli M, Bartoli D, Pieroni S, Pettirossi V, Piobbico D, Viola Magni M, Servillo G. HOPS: a novel cAMP-dependent shuttling protein involved in protein synthesis regulation. J Cell Sci 2005; 118:3185-94. 1466 Cell Cycle 2008; Vol. 7 Issue 10