CIP1 and contributes to

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Nucleophosmin/B23 interacts with p21WAF1/CIP1 and contributes to its stability

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Jianyong Xiao,1-3,† Zhiyi Zhang,1,2,† George Gong Chen,4 Meifang Zhang,1,2 Yuanjie Ding,1,2 Jia Fu,1,2 Mingtao Li3 and Jing-Ping Yun1,2,* of Pathology; Cancer Center of Sun Yat-sen University; Guangzhou, China; 2State Key Laboratory of Oncology in Southern China; 3Department of Pharmacology; Zhongshan School of Medicine; Sun Yat-sen University; Guangzhou, China; 4Department of Surgery; Faculty of Medicine; The Chinese University of Hong Kong; Hong Kong, China †These

authors contributed equally to this work.

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Abbreviations: NPM, nucleophosmin; Act D, actinomycin D; CHX, cycloheximide; IP, immunoprecipitation; IB, immunoblotting

p21 is a short-lived protein, and proteasome inhibition leads to an increase in p21 protein levels and an increase in p21 half life.4 Basal turnover of p21 controlled by proteasome does not appear to require ubiquitination, although a role for N-terminal ubiquitination of p21 has been proposed.5,6 TSG101 protein binds to p21 and increases the stability of the p21 protein in HEK293F cells and differentiating primary keratinocytes.7 Recently, WISp39, an hsp90-binding TPR protein, was shown to interact with p21 and regulate its stability.8 Nucleophosmin (NPM, B23, numatrin, NO38) is a nucleolar phosphoprotein constantly shuttling between the nucleolus and cytoplasm.9 It functions as a ribosomal assembly and transport protein; it binds to pRb and synergistically stimulates DNA polymerase α;10 it is essential in centrosome duplication;11 and it targets proteins such as GADD45α and p120 by nuclear localization signals (NoLS) for their import.12,13 Nucleolar translocation of NPM occurs in mitosis as the nucleolus disappears in the prophase.14 NPM is redistributed from the nucleolus in response to cytotoxic drugs such as Actinomycin D and genotoxic stress.15 It has been demonstrated to interact with a few short-lived proteins such as ARF and Bax acting as a chaperone.16,17 NPM contains an N-terminal oligomerization domain that performs chaperone activities such as preventing protein aggregation, protecting enzymes during thermal denaturation and facilitating renaturation of chemically denatured proteins.18 The association of NPM with p53 regulates the stability and activity of p53.19 Depending on the expression levels and gene dosage, NPM seems to function as either an oncogene or a tumor suppressor.20 The aim of the present study was to help elucidate the mechanism of p21 stabilization at the post-translational level. We hypothesized that NPM might be a positive regulator of p21. Indeed, we found that NPM physically interacted with p21, and it can inhibit p21 ubiquitination and prolonged its half life.

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The cyclin-dependent kinase inhibitor p21WAF1/CIP1 is a critical regulator of cell cycle, and it is easily degraded by proteasome through ubiquitin-dependent and -independent pathway. The mechanism of the post-translational regulation of p21 stability remains to be further clarified. In the present study, we have identified nucleophosmin (NPM)/B23, a multifunctional protein that bound p21 and contributed to its stability. The direct interaction between p21 and NPM was confirmed by reciprocal co-immunoprecipitation and GST pull-down assay. Confocal microscopy showed that NPM partially co-localized with p21 in nucleoplasm and their co-localization increased treated with Act D which induces the nucleoplasmic translocation of NPM. We observed the half life of p21 was prolonged with overexpression of NPM or Act D treatment. Knockdown of NPM by siRNA resulted in downregulation of p21 and impaired upregulation of p21 treated with Act D. Further, we examined the effect of NPM expression on the ubiquitination of p21. Overexpression of NPM inhibited the ubiquitination of p21, and depletion of NPM remarkably improved the ubiquitination of p21. Altogether, we provide evidence for a direct binding between NPM and p21, and assign NPM as a positive post-translational regulator of p21.

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Key words: NPM/B23, p21, interaction, half life, ubiquitination, stability, actinomycin D

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p21 (Waf1/Cip1), which belongs to the Cip/Kip family of cyclindependent kinase inhibitors, has important roles in cell proliferation, differentiation, senescence and apoptosis.1 Transcriptional control of p21 by both p53-dependent and -independent mechanisms is critical for growth arrest.2 At the post-translational level, p21 has been reported to be regulated by modifications such as phosphorylation.3 *Correspondence to: Jing-Ping Yun; Department of Pathology; Cancer Center; Sun Yat-sen University; No. 651, East Dongfeng Road; Guangzhou 510060 China; Tel.: 8620.87343602 ext. 815; Email: [email protected] Submitted: 12/15/08; Revised: 01/11/09; Accepted: 01/21/09 Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/article/7898 www.landesbioscience.com

Results NPM directly interacts with p21 through its amino acids 117–187. First, we investigated whether complexes between

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Figure 1. NPM physically interacts with p21, in vivo and in vitro. (A) Cell lysate from HepG2 cells was immunoprecipitated with anti-p21 (71-1000, Zymed Laboratories, CA) or IgG (negative control). Immunoprecipitates were resolved by SDS-PAGE, transferred to PVDF membrane, and then probed with antip21 (556430, BD Pharmingen, CA) and anti-NPM antibodies. (B) HEK293T cells were transfected with pFLAG-p21 for 24 hrs. Cell lysate was subjected to immunoprecipitation with antibodies against FLAG or NPM. The resulting immune-complex was then analyzed by immunoblotting. (C) HEK293T cells were co-transfected with pFLAG-p21 and pHA-NPM for 24 hrs. Cell lysate was subjected to immunoprecipitation with antibodies against HA, followed by immunoblotting. (D) Top, the domain structure of the full-length His-NPM protein and the various deletion mutants. Bottom, identification of the various NPM deletion mutants critical for p21 binding. Bacterially purified GST-p21 proteins were mixed with bacterially purified His-NPM or the His-NPM deletion mutants. The reactive samples were subjected to precipitation using GST affinity beads and immunoblotted with anti-GST and anti-His antibodies. His recombinant proteins were verified (left 1–6 lanes).

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e­ ndogenous NPM and p21 could be detected in vivo. As seen in Figure 1A, NPM was present in p21 immunoprecipitates, but not in normal rabbit IgG precipitates. To confirm the interaction of NPM and p21 in vivo, plasmids harboring FLAG-tagged p21 (pFLAG-p21) and HA-tagged NPM (pHA-NPM) were constructed. HEK293T cells were transfected with expression vectors for pFLAG-p21, or pFLAG-p21 plus pHA-NPM. Cell lysate was analyzed by immunoprecipitation with antibodies against FLAG and HA. Immunoblotting analysis revealed the binding of exogenous FLAG-p21 and endogenous NPM (Fig. 1B) and the interactions between ectopically produced FLAG-p21 and HA-NPM (Fig. 1C). 890

To further determine which functional domain of NPM was required for the interaction with p21, we performed a binding assay utilizing bacterially purified NPM and p21 in vitro. A series of His-NPM deletion mutants that covered distinct functional domains of NPM (illustrated in Fig. 1D, top) were generated: His-F1R1 (full length NPM), His-F1R3 (amino acids 1–117), His-F1R4 (amino acids 1–187), His-F1R5 (amino acids 1–259) and His-F2R1 (amino acids 118–295). Wild-type NPM and the four mutants were incubated with GST-p21 fusion protein as well as GST tag as a negative control, and precipitated with GST affinity beads. The precipitates were immunoblotted for His and GST (Fig. 1D, bottom). The pull-down assays indicated that the domain (amino acids 117–187)

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NPM inhibits the ubiquitination of p21. p21 can be degraded by proteasome in a ubiquitin-dependent manner.21 Thus, we wanted to examine the effect of NPM on the ubiquitination of p21 in vivo. First, we checked the ubiquitination level of p21 in response to Act D treatment. As shown in Figure 4A, the effect of Act D was time dependent with the ubiquitination level of p21 being greatly reduced 24 hrs after Act D treatment. To test whether the inhibition of ubiquitination by Act D is p21 specific or universal, we blotted the total cell lysate with an anti-ubiquitin antibody. There was no obvious change Figure 2. The co-localization of NPM and p21 in HepG2 cells. HepG2 cells were treated with Act D (0.05 in Act D-treated cells compared with μg/ml) for 24 hrs, fixed and then immunostained with antibody against NPM or p21 followed by treatment untreated cells (data not shown). To with fluorescein isothiocyanate-conjugated goat anti-mouse IgG or cy3-conjugated goat anti-rabbit IgG. Cell nuclei were visualized by Hoechst 33258 staining. Immunofluorescence patterns were recorded with further confirm that Act D influenced confocal laser scanning microscopy. The label “Merge” indicates the combined images of the two proteins the p21 ubiquitination through the in the nucleus. B23-p21 pathway, we employed siRNA to knockdown the expression of NPM. of NPM may be essential for the direct association between NPM The data showed that the ubiquitination of p21 was partially rescued and p21. by the knockdown of NPM in Act D-treated cells, which suggested NPM partially co-localizes with p21. NPM mainly localizes that NPM participated in the regulation of p21 ubiquitination under GC region of nucleolus with a smaller nuclear diffuse fraction in treatment Act D (Fig. 4B). To corroborate that NPM negatively regunormal conditions, whereas p21 is a mostly nuclear diffuse protein lates the ubiquitination of p21 in vivo, we transiently overexpressed that is excluded from the nucleolus. Accordingly, staining of HepG2 Myc tagged NPM protein in cells. As seen in Figure 4C, a high level cells with antibodies against p21 and against NPM indicated only a of NPM decreased the ubiquitination of p21. After knocking down small degree of co-localization of the two proteins. Act D treatment, the expression of NPM using siRNA, the ubiquitination of p21 was however, induced a remarkable nuclear redistribution of NPM, markedly increased in comparison with the negative control (Fig. 4D). The results indicated that NPM negatively regulated the ubiqwhich showed an increased co-localization with p21 (Fig. 2). NPM stabilizes p21 protein level. We next tested the effect of uitination of p21. NPM depletion on the expression level of p21 in HepG2 cells under basal conditions and after Act D treatment. Under basal conditions, Discussion knockdown of NPM using siRNA decreased the protein level of p21 is a critical cell cycle regulator, but it is an unstable protein p21 (Fig. 3A). After Act D treatment, the expression level of p21 that is degraded by the proteasome pathway. In the present study, we increased. Notably, the upregulation of p21 treated with Act D was identified NPM, a multiple-functional shuttle protein, as a positive abrogated by transfection with NPM siRNA (Fig. 3B). These data regulator of p21 that binds with p21 and inhibits the ubiquitination indicate NPM positively regulates p21 protein level. of p21. These results provide evidence for a previously unknown Unlike short-lived p21 protein, NPM is a very stable protein and mechanism by which p21 is stabilized. usually functions as a chaperone of target proteins such as ARF and Our results suggest that post-translational regulation plays Bax.16,17 On the basis of the interaction between NPM and p21, an important role in p21 stability. Several studies have reported we posited NPM might regulate p21 level at post-translational level. post-translational regulation of p21 stability. Since p21 protein is Therefore, we examined the effect of NPM on the half life of p21 unstructured when expressed in bacteria,22 it has been suggested that the binding partners of p21 determine p21 stability, although using CHX (Cycloheximide, a protein synthesis inhibitor). At first, we checked the half life of p21 after Act D treatment or the underlying mechanisms remained to be determined. p21 lacking in combination with NPM silence. As shown in Figure 3C, the half the cdk-cyclin binding site is unstable whereas the p21 lacking the 23 life of p21 was significantly greater under Act D stimulation. After PCNA binding site is more stable. In another report, cyclin D1 24 depletion of NPM, Act D-treated cells showed no obvious variations was shown to inhibit p21 degradation by the 20S proteasome. Several phospho-site-deficient or phospho-site-mimicking mutants in p21 half life comparing with Act D-untreated cells. The results of p21 have been proposed to regulate p21 stability by modulating suggest that Act D treatment leads to longer half life of p21 via NPM the interaction with p21 binding proteins or by translocating p21 pathway. To further confirm that NPM positively affects the half life to the cytoplasm. These studies have given some contradictory of p21, we upregulated the expression level of NPM with transient results, probably due to problems associated with overexpression of transfection of pMyc-NPM. As seen in Figure 3D, the half life of p21 p21 vectors resulting in the saturation of the degradation pathway.8 was increased by overexpression of NPM. The results indicated that In addition, TSG101 protein binds to p21 and increases stability NPM prolongs the half life of p21. of the p21 protein in HEK293F cells and differentiating primary www.landesbioscience.com

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Figure 3. NPM positively impacts the half life of p21. (A) HepG2 cells were transfected with NPM siRNA or Ctrl siRNA for 72 hrs followed by immunoblotting using antibody against NPM and antibody against p21 (556430, BD Pharmingen, CA). (B) HepG2 cells were transfected with NPM siRNA or Ctrl siRNA for 48 hrs and irradiated with Act D (0.05 μg/ml) for additional 24 hrs. Immunoblotting was done using antibody against NPM and antibody against p21 (556430, BD Pharmingen, CA). (C) The knockdown of NPM impairs the Act D-induced longer half life of p21. HEK293T cells were transfected with pcDNA3.1-p21 or with a combination of NPM siRNA or Ctrl siRNA and treated with Act D (0.05 μg/ml) for 24 hrs. Cell lysate was prepared at the indicated time points after addition of CHX (20 μg/ml) and then immunoblotted with anti-p21 and anti-Actin antibodies. (B) Overexpression of NPM retards the degradation of p21. 293T cells were co-transfected with pcDNA3.1-p21 and either pMyc-NPM or pCMV-Myc. Cell lysate was prepared at the indicated time points after the addition of CHX and then immunoblotted with anti-p21 and anti-Actin antibodies.

keratinocytes. However, TSG101 does not function as a general determinant of p21 stability in keratinocytes, but it counteracts selectively the destabilization of the p21 protein that accompanies differentiation.7 Recently, WISp39 was described as a p21 binding 892

partner that regulates p21 stability in vivo. The siRNA-mediated downregulation of WISp39 decreases the half life of endogenous p21.8 However, the authors did not provide data on how WISp39 might stabilize p21.

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Figure 4. NPM decreases the ubiquitination of p21. (A) HEK293T cells transfected with pFLAG-p21 or pHA-Ub or with combinations of these vectors were treated with Act D (0.05 μg/ml) for the indicated times. Cell lysate was prepared at 6 hrs after MG132 (20 μM) treatment, immunoprecipitated with antiFLAG antibody and immunoblotted with anti-HA antibody (to detect ubiquitin-conjugated proteins) or with anti-FLAG antibody. (B) The depletion of NPM partially rescues the reduced ubiquitination of p21 under Act D treatment. HEK293T cells transfected with pFLAG-p21, pHA-Ub, NPM siRNA or Ctrl siRNA were treated with Act D (0.05 μg/ml) for 24 hrs. Cell lysate was prepared at 6 hrs after MG132 (20 μM) treatment and immunoprecipitated with anti-FLAG antibody. Immunoblotting was performed using anti-HA antibody, anti-FLAG antibody and anti-NPM. (C) Overexpression of NPM impairs the ubiquitination of p21. HEK293T cells transfected with pFLAG-p21, pHA-Ub, pMyc-NPM or pCMV-Myc were incubated with MG132 (20 μM) for 6 hrs. Cell lysate was immunoprecipitated with anti-FLAG antibody, then immunoblotted with anti-HA antibody, anti-FLAG antibody and anti-Myc. (D) Knockdown of NPM increases ubiquitination of p21. HEK293T cells transfected with pFLAG-p21, pHA-Ub, NPM siRNA or Ctrl siRNA was treated with MG132 (20 μM) for 6 hrs. Cell lysate was immunoprecipitated with anti-FLAG antibody. Immunoblotting was done with anti-HA antibody, anti-FLAG antibody and anti-NPM.

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Our data show that the expression of NPM negatively affects the ubiquitination of p21 in vivo. NPM may bind to the N-terminus of p21 and block its ubiquitination and subsequent proteasomal degradation because the site of N terminal lysine residues is critical in making p21 ubiquitination. This may be the mechanism by which NPM increases the half life of p21. Even though we mainly focused our study on the ubiquitin-dependent way, NPM may also influence the stability of p21 via an ubiquitin-independent way. MDM2 promotes p21 proteasomal turnover independently of ­ubiquitination.25 Previously, NPM was reported to impair MDM2-p53 interaction and stabilize p53.26 An attractive possibility is that NPM might stabilize p21 in www.landesbioscience.com

a same manner by abrogation of MDM2-p21 interaction. These hypotheses need to be further studied. Our results provide evidence that NPM (amino acids 117–187) directly binds to p21. NPM has both chaperone and nucleuscytoplasm shuttling properties, through which it is thought to prevent protein aggregation in the nucleolus during ribosome assembly and to facilitate the transport of ribosomal proteins. Several unstable proteins such as p53 and ARF also directly bind to NPM. NPM exhibits chaperone ability in regulating the stability and ­transcriptional activity of p53 and in protecting ARF from degradation. NPM might function similarly as molecular chaperone for

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anti-His (34660, Qiagen, Hilden, Germany), polyclonal anti-HA (sc-805, Santa Cruz Biotechnology, CA, USA), polyclonal anti-GST (sc-459, Santa Cruz Biotechnology, CA, USA), monoclonal anti-Myc (AB-103-10, Tiangen Biotech, Beijing, China) and monoclonal antiActin (ms-1295-po, NeoMarkers, CA, USA). Cell culture and transfection. HepG2 and HEK293T cell lines were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml of penicillin and streptomycin. For transfection, 5 x 105 cells were seeded into 6-well plates the day before transfection. In each well, 4 μg of DNA and 10 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) were added in 250 μl of Opti-MEM in separate tubes. Solutions were mixed gently, allowed to stand for 20 min at room temperature and then added to each well at 37°C. After 8 hrs, 2 ml fresh DMEM medium was added, and the plates were incubated in a CO2 incubator. RNA interference. Small interfering RNA for NPM (NPM siRNA) and nonspecific siRNA (Ctrl siRNA) were synthesized as UGA UGA AAA UGA GCA CCA G and GUU CUC CGA ACG UGU CAC G, respectively.19 For RNA interference, HepG2 cells were added to 6-well plates 24 hrs prior to transfection and grown to 2 x 105. For each well, 100 nM siRNA was transfected into cells with Lipofectamine 2000 according to the manufacturer’s protocol. After 48–72 hrs, the cells were ready for gene knockdown analysis. Actinomycin D and Cycloheximide treatment. Cells in log growth phase were exposed to a certain concentration of Actinomycin D (Act D, 0.05 μg/ml) or Cycloheximide (CHX, 20 μg/ml) for varying times. Cells were then lysed in RIPA lysis buffer [0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% NP-40, 10 mM EDTA] (20-188, Upstate, CA, USA) supplemented with 1 mM sodium orthovanadate, 1 mM sodium fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin and 1 mM PMSF (70153020, Roche, Germany). Immunoprecipitation and immunoblotting. For immunoprecipitation (IP), cells were lysed in RIPA buffer supplemented with proteinase inhibitor cocktail. Specific antibodies were added for 2 to 3 hrs, and then protein A/G beads were added for an additional 2 hrs. We dissolved the precipitated proteins in 3x sodium dodecyl sulfate (SDS) loading buffer and fractionated them by SDS-polyacrylamide gel electrophoresis (PAGE). For immunoblotting (IB), cellular lysate or immunoprecipitation complex were boiled with 3x SDS loading buffer and then fractionated by SDS-PAGE. The proteins were transferred to PVDF membrane, which was then incubated with primary specific antibody in 5% milk, followed by a horse radish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit antibodies and ECL detection reagent (Amersham Life Science, Piscataway, NJ, USA). GST pull-down assays. GST-p21 and His-NPM (wild-type and mutant) proteins were incubated in phosphate-buffered saline (PBS) for 3 hrs at 4°C. GST affinity beads were added, and the proteins were incubated for an additional 2 hrs. After extensive washes with the RIPA buffer, the precipitates were resolved by SDS-PAGE and subjected to immunoblotting analysis. Immunofluorescence. Cells grown on cover slips were fixed for 20 min in PBS containing 4% parafomaldehyde, permeabilized in 0.1% Triton X-100 for 2 x 5 min and incubated in blocking buffer (3% horse serum in TBST) for 1 hr. Cells were incubated in ­antibody dilution buffer (3% bovine serum albumin in TBS) containing the indicated primary antibody for 2 hrs in room temperature and then

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p21, preventing self-aggregation and aiding protein complex formation. Indeed, interactions between chaperones and their substrates are highly dynamic and typically have low stoichiometry, which explains partial nuclear co-localization of NPM and p21 observed by confocal microscopy. Our data indicate that NPM is also required to stabilize p21 in HEK293T cells after Act D treatment, suggesting that Act D can induce increase of p21 protein independent of p53. Previous studies reported that Act D treatment causes p53-dependent upregulation of p21 protein in tumor cells.27,28 It is likely that that Act D induces the high expression of p21 via both p53-dependent and -independent pathways. Similarly, standard DNA damaging chemotherapeutic agents, such as Adriamycin, increase p21 through p53-dependent and -independent pathways.29 Expression of p21 may result in a universal inhibition of cyclin-dependent kinase activities, and induce diverse cell cycle phenotype.30 We hypothesized that NPM gets involved in cell cycle regulation via p21. We observed the effect of NPM depletion on the cell cycle progression of HepG2 cells under basal conditions and after Act D treatment. Under basal conditions, transfection with NPM siRNA caused a small and consistent increase in the proportion of S phase cells comparing with negative control (Fig. S1A). After Act D treatment, HepG2 cells treated with control siRNA accumulated in G2/M phase. In contrast, NPM-depleted cells failed to arrest in G2/M phase, and the percentage of cells in S phase was significantly higher than in control cells (Fig. S1B). Notably, similar cell cycle profiles have been described in response to X-ray irradiation for U2OS and p53+/- cells.19,31,32 Furthermore we transfected FLAG tagged p21 expression plasmid into NPM-depleted HepG2 cells. However, overexpression of p21 failed to rescue Act D-induced S arrest in NPM-depleted HepG2 cells (data not shown), suggesting multiple pathways through which NPM regulates cell cycle in HepG2 cells. In summary, we identified NPM as a protein that targets p21 to stabilize it. This is a novel mechanism by which p21 is regulated at the post-translational level.

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Plasmids and antibodies. pcDNA3.1-p21 and pGST-p21 were constructed by inserting p21 cDNA into the pCDNA3.1 and pGEX-4T-1 vectors, respectively. pEGFP-NPM and pMyc-NPM were constructed by inserting NPM cDNA into the pEGFP-C1 and pCMV-Myc vectors, respectively. pCDNA3.1-HA was constructed by inserting 3x HA into the opening read fragment of pCDNA3.1 vectors. pHA-NPM was generated by double restriction enzyme excision of NPM from pEGFP-NPM and ligation to pcDNA3.1-HA. In order to generate pHis-NPM constructs, regions encoding full-length NPM (F1R1), amino acids 1–117 (F1R5), 1–187 (F1R3), 1–259 (F1R4) and 117–295 (F2R1) were amplified by PCR. The PCR products were cloned into pET28a vector. The pFLAG-p21 expression vector was kindly provided by Dr. Shimei Zhuang (Sun Yat-sen University, China). The HA-tagged ubiquitin construct (pHA-Ub) was a kind gift from Dr. Dirk Bohmann (University of Rochester, NY). The antibodies used in this study were as follows: monoclonal anti-NPM,33 polyclonal anti-p21 (71-1000, Zymed Laboratories, CA, USA), monoclonal anti-p21 (556430, BD Pharmingen, CA, USA), monoclonal anti-FLAG (M2, Sigma, USA), monoclonal 894

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We thank Shimei Zhuang (Sun Yat-sen University, China) for kindly providing the pFLAG-p21 vector, Dr. Dirk Bohmann (University of Rochester, NY) for the kind gift of pHA-Ub, and Jinke Cheng (Shanghai Jiao Tong University, China), Xinyuan Guan (University of Hong Kong), Musheng Zeng (State Key Laboratory of Oncology in Southern China) for helpful discussions. This work was supported by National Natural Science Foundation of China (No. 30471960 and No. 30772492), Guangdong Natural Science Foundation (No. 06021198 and No. 8151008901000057) and in part by the National High Technology Research and Development Program of China (863 Program) (No. 2006AA02A404). Supplementary materials can be found at: www.landesbioscience.com/supplement/XiaoCC8-6-Sup.pdf References

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Acknowledgements

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washed extensively in PBS before being incubated with the appropriate fluorochrome-conjugated secondary antibody for 1 hr. DNA was stained by Hoechst. We mounted stained cells on glass slides and examined them by confocal microscopy.

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