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1 Department of Genetics, B-3, Graduate School of Medicine, and 2 Graduate School of Frontier Biosciences, Osaka University, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
3 Solution-Oriented Research for Science and Technology, Japan Science and Technology Agency, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
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Introduction |
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, and also with the transcriptional coactivators p300 and CREB-binding protein (CBP) (Hsu et al. 2001; Hirose et al. 2003). SEI-1 (TRIP-Br1) and TRIP-Br2 possess an intrinsic transactivation activity and regulate the transcriptional activity of E2F-1 via interaction with DP-1, an E2F-1 partner protein (Hsu et al. 2001). These features imply that SEI-1 and TRIP-Br2 play a dual role in the control of cell-cycle progression through the transcriptional regulation of E2F-responsive genes and a regulatory interaction with the CDK4/cyclinD complex.
In mammals, two more SEI family proteins, hematopoietic progenitor protein (Hepp) and replication protein-binding transactivator 1 (RBT1), have been identified (Cho et al. 2000; Abdullah et al. 2001). Although Hepp has not been functionally characterized, RBT1 was shown to bind to the second subunit of replication protein A (RPA32) and is thus implicated in DNA replication, repair, or recombination. Recently, a genetic screen for homeotic gene modifiers in Drosophila identified a gene, taranis (tara), whose products (TARA- and -ß) contain a 
40-amino acid motif that is specifically conserved in human SEI-1, and the related proteins TRIP-Br2, Hepp, and RBT1 (Calgaro et al. 2002). In addition to this characteristic motif, designated as SERTA motif (for SEI-1, RBT1, and TARA), there are three other regions conserved in these human and Drosophila proteins, suggesting that the SEI family proteins have a shared biological function. A genetic analysis suggested that tara is a novel member of the trithorax group (TrxG) gene, and its products play a role in the maintenance and/or remodeling of chromatin structure to regulate the transcription of the TrxG target genes (Calgaro et al. 2002).
The fact that SEI-1 interacts with E2F-1/DP-1 and CBP raises the possibility that SEI family proteins may regulate other transcription factors and/or chromatin-remodeling proteins involved in cell-cycle control. The p53 tumor suppressor protein is activated by cellular stresses, including DNA damage, oncogene activation, and hypoxia, and p53 thus activated regulates cell-cycle arrest and apoptosis via transcriptional activation of many target genes such as p21, 14-3-3 
, PUMA, and BAX (Vousden & Lu 2002; Vogelstein & Kinzler 2004). p53 activity is controlled by post-translational modifications, including phosphorylation, ubiquitination, and acetylation, and also by interactions with various p53 regulators (Vousden & Lu 2002; Brooks & Gu 2003). For example the cellular level of p53 is negatively regulated by the Mdm2 oncoprotein, and by E6-associated protein (E6AP) in conjunction with the human papillomavirus (HPV) E6 oncoprotein. Furthermore the transactivating function of p53 is regulated also by interaction with transcriptional regulators such as CBP, p300, Sir2, ING, ASPP (Luo et al. 2001; Samuels-Lev et al. 2001; Vaziri et al. 2001; Feng et al. 2002; Vousden & Lu 2002; Brooks & Gu 2003).
Here we present evidence that human SEI family proteins regulate p53-dependent transcriptional activation and that the over-expression of SEI members causes p53-independent growth inhibition. Our results, together with earlier studies, suggest that SEI family nuclear factors play a role in cell-cycle regulation through interactions with transcription factors and/or chromatin-remodeling machineries.
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Results |
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Figure 1A shows a schematic representation of the three SEI proteins investigated in this study. Although the SEI family proteins were originally designated by different names, we use a simple terminology that follows from the name of the first-described SEI member, p34SEI-1 (Sugimoto et al. 1999), to designate the members of this family: SEI-1 (TRIP-Br1), SEI-2 (TRIP-Br2 or Y127), and SEI-3 (Hepp). All the SEI proteins contain four characteristic sequence motifs: N-terminal nuclear localization signal (NLS)-like, SERTA, PHD-bromo-binding, and C-terminal motifs (Sugimoto et al. 1999; Hsu et al. 2001; Calgaro et al. 2002). We examined the expression of the SEI mRNAs in mouse tissues and human cell lines by quantitative RT-PCR (Fig. 1B,C). The SEI-1 and SEI-2 mRNAs were expressed at a similar level in most of the mouse tissues tested, except for the liver. In contrast, SEI-3 mRNA was expressed strongly in the thymus, spleen, and bone marrow. The human cell lines HeLa, MCF7, and U2OS cells expressed all three SEI mRNAs, although the expression of the SEI-2 mRNA was relatively low.
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To investigate whether SEI proteins are involved in the regulation of the p53 signaling pathway, we examined the effect of their increased expression on the transactivation function of p53 by luciferase reporter assays. HeLa cells were co-transfected with a p53 reporter plasmid containing tandem p53-binding sites in its promoter region (p53-Luc) and an expression plasmid encoding a Flag-tagged human SEI protein. As shown in Fig. 2A, little basal expression of the luciferase reporter was observed when the cells were co-transfected with the vector plasmid (pEF-BOS-EX). In contrast, co-expression with either SEI-1 or SEI-2 strongly enhanced the p53 reporter activity. SEI-3 also significantly stimulated the reporter activation, but an unrelated protein, Rad9, did not. The activation of the p53 reporter gene was dependent on the dosage of the transfected SEI-expression plasmids; of them, SEI-2 was most effective (Fig. 2B). Since the endogenous p53 protein level in HeLa cells is known to be low because of the p53-degrading function of the HPV E6 protein, which is expressed in this cell line (Talis et al. 1998; Vogelstein & Kinzler 2004), we tested the effect of expressing exogenous p53 (Fig. 2C). Transfection with a p53 expression plasmid increased the basal reporter activity (Vector), which was further activated by the co-expression of SEI proteins. On the other hand, expression of the HPV-16 E6 protein strongly inhibited the effect of the SEI proteins (Fig. 2D), suggesting that SEI and E6 regulate p53 function in a mutually opposing manner.
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To address whether the SEI proteins specifically regulate the transcriptional activation of p53, other reporter constructs, driven by the E2F-responsive promoter, actin promoter, or elongation factor 1 promoter, were tested. As shown in Fig. 3A, untagged mouse SEI proteins as well as Flag-tagged human SEI proteins strongly activated the expression of the p53-Luc reporter, up to 100-fold, but showed little effect (< 2.3-fold) on the luciferase expression of the other reporter genes. The SEI-1 and SEI-2 proteins also stimulated the transactivation function of p53 in osteosarcoma U2OS cells (Fig. 3B). In contrast, none of the SEI proteins activated the p53 reporter gene in the p53-deficient human osteosarcoma Saos2 cells. To further examine whether the SEI-dependent reporter activation was mediated by p53, we established a p53-knockdown HeLa cell line (HG18 cells) by stable expression of a p53-specific short hairpin RNA (shRNA) (Brummelkamp et al. 2002). The p53 protein level was strongly reduced in HG18 cells, where the reporter expression was not stimulated by any SEI protein (Fig. 3C). Co-expression of p53 in HG18 cells rescued the effect of the SEI proteins on the reporter activation (HG18 +p53), indicating that the SEI proteins require p53 for this transactivation function. These results indicate that SEI proteins specifically stimulate the transactivation activity of wild-type p53 protein.
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To learn more about SEI functions, we investigated whether the proteins possessed an intrinsic transactivation activity. When fusion proteins of Gal4 DNA-binding domain and SEI proteins (Gal4-SEIs) were tested using a reporter gene containing Gal4-binding sites, the Gal4 fusions of SEI-1 and SEI-2 strongly activated the reporter gene transcription (2000-fold) whereas Gal4-SEI-3 showed a weak but significant (16-fold) activity (Fig. 4A). The reporter activation by these Gal4-SEI fusions was comparable in the p53-knockdown HG18 cells, and was not affected by the co-expression of the wild-type or mutant p53, indicating that the intrinsic transactivation activity of SEI proteins is independent of the p53 protein.
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Previous studies showed that SEI-1 and SEI-2 interact with the PHD and/or CH3 region of CBP (Hsu et al. 2001; Hirose et al. 2003). Consistent with these reports, we found that CBP was co-immunoprecipitated with the SEI proteins from cell lysates when it was co-expressed with them in COS7 cells (Fig. 4C). We also looked for a physical interaction between the SEIs and INGs since ING proteins contain a PHD finger motif, but we did not observe their interaction by co-immunoprecipitation (not shown).
SEI proteins stimulate the p53-p21 signaling pathways
We next examined the effect of inducible expression of SEI proteins on p53 function by using the reverse tetracycline (Tc)-controlled transactivator (rtTA) system. HeLa-Tet-On (HAM3) cells and U2OS-Tet-On cells were co-transfected with plasmids containing each SEI-Flag coding sequence under the Tc-responsive promoter and a hyg-selection plasmid, and then the hyg-resistant clones were isolated. Figure 5A shows that the expression of Flag-tagged SEI-1, SEI-2, and SEI-3 proteins, with respective sizes of 34, 43, and 33 kDa, was induced by doxycycline (Dox) treatment of the HAM3- or U2OS-Tet-On-derived clones, although the expression level of SEI-2 was consistently lower than those of other SEIs in any clones we tested. Immunofluorescence staining of Dox-treated U2OS transformants revealed that all of the SEI proteins were localized to the nucleus, and a minor fraction of SEI-1 and SEI-3 was present in the cytoplasm (data not shown). We examined whether the mRNA or protein levels of p53 and its target p21 were regulated by the SEI proteins. A Northern blot analysis of the HeLa-derived H4.8 cells revealed that Dox-induced expression of SEI-1 protein resulted in strong expression of p21 mRNA without a significant increase in the p53 mRNA level (Fig. 5B). On the other hand, an immunoblot analysis showed that inducing the expression of SEI-1 strongly up-regulated the protein levels of both p53 and p21 (Fig. 5C), indicating that SEI-1 does not enhance the transcription of p53 mRNA but may regulate the stability of p53 protein. In contrast, Dox-induced expression of SEI-2 or SEI-3 in the HeLa-derived cells resulted in only weak increases in protein levels of p53 and p21, although the mRNA levels of p53 are rather up-regulated slightly (Fig. 5B,C). In the U2OS-derived cells, SEI-1 and SEI-2 clearly up-regulated the p53 and p21 protein levels, whereas SEI-3 had little effect (Fig. 5D). These results collectively suggest that the SEI proteins are unlikely to regulate p53 expression at the transcriptional level, and that some of them up-regulate the p53 activity probably by protein stabilization of p53, which in turn activates the p21 gene.
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We next examined the effect of inducible expression of SEI proteins on cell growth. As shown in Fig. 6A,B, the Dox-induced expression of SEI proteins resulted in strong growth inhibition of HeLa and U2OS-derived clones. The treatment of control cells (Vector) with Dox did not affect their growth. However, the growth-inhibitory activity of individual SEI proteins did not necessarily correlate with the increase in the p53 or p21 protein levels. For example, expression of SEI-2 caused strong growth inhibition, but resulted in a slight increase in the level of p53 or p21 in HeLa cells (Figs 5C and 6A). To test whether the SEI-induced growth inhibition was mediated by p53, we established p53-knockdown cells from the HAM3-derived clones, H4.8, H3.4, and H2.4, by transfection with the p53-shRNA expression plasmid. As shown in Fig. 6C, the resultant clones did not express a detectable level of p53 even after Dox treatment, which potentially up-regulated the p53 protein level in SEI-1 (H4.8) cells (Fig. 5C). As shown in Fig. 6D, however, the Dox-induced expression of SEI proteins inhibited the cell growth of these p53-knockdown clones as strongly as their respective control clones (VC). This result suggests that p53 is not required for the SEI-induced growth inhibition of HeLa cells and that over-expression of the SEIs may activate a p53-independent pathway leading to growth inhibition.
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Discussion |
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Our findings, together with those of previous studies, have revealed several aspects of the nuclear functions of SEI family proteins. First, SEI proteins show an intrinsic transactivation activity when fused to a DNA-binding domain. Their strong transactivation function was mapped to a relatively short (60–80 amino acids) C-terminal region (Cho et al. 2000; Hsu et al. 2001), suggesting that the transactivating function may be fulfillled through interaction with other proteins. Second, SEI proteins are present mostly in the nucleus and can interact with bona fide coactivators or corepressors such as CBP, p300, TIF1, and KRIP-1 (TIF1ß), and with transcriptional modulators such as DP-1 and HPV-E6 (Hsu et al. 2001; Gupta et al. 2003; Hirose et al. 2003). Third, the SEIs’ ability to enhance p53's transactivation function is itself cooperatively enhanced by the ING1b and ING2 proteins, which are implicated in chromatin remodeling and the regulation of histone acetylation or deacetylation. Fourth, a novel TrxG gene in Drosophila, taranis, encodes a SEI family protein that cooperates with other TrxG members and antagonizes Polycomb group (PcG) members, probably by modifying the chromatin structure (Calgaro et al. 2002). Taken together, these observations strongly suggest that the SEI family nuclear factors play a role in transcriptional regulation as chromatin modulators that physically and functionally interact with coactivators and/or corepressors.
The SEI proteins have been suggested to regulate cell-cycle progression via interactions with CDK4/cyclinD and E2F1/DP-1 (Sugimoto et al. 1999; Hsu et al. 2001; Li et al. 2004). We found that the induced expression of SEI proteins inhibited cell growth of HeLa and U2OS cells. We had initially postulated that the SEI-induced growth arrest might be mediated by p53. However, the siRNA-mediated knockdown of the endogenous p53 protein in HeLa cells did not alter the SEI-induced growth arrest, indicating that the SEI members can activate a growth-inhibitory pathway that is independent of p53 function. We have not identified which molecules are involved in this pathway, but plausible candidates would be CDK4/cyclinD and E2F1/DP-1 complexes, which may regulate cell-cycle progression independently of p53 activity.
In summary, we have shown that SEI family nuclear factors strongly stimulate the transactivating function of p53, probably via their interaction with CBP, and that they induce p53-independent growth inhibition when over-expressed. Although we have not examined whether the SEI proteins play an essential role in p53-induced gene expression or in the cellular functions of p53, this important issue is to be elucidated by siRNA-mediated knockdown of endogenous SEI proteins in the future studies. Furthermore, an expression array analysis of SEI-regulated genes and an interaction screen for other binding partners of SEI proteins would be necessary to elucidate the precise molecular mechanism underlying the SEI-dependent transcriptional regulation.
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Experimental procedures |
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Human cDNAs for SEI-1, SEI-3, Rad9, ING1b, and ING2, and mouse cDNAs for SEI-1, SEI-2, and SEI-3 were obtained by reverse transcriptase-primed (RT-) polymerase chain reaction (PCR). Human SEI-2 cDNA (KIAA0127) (Nagase et al. 1995) was provided by Dr Takahiro Nagase (Kazusa DNA Research Institute, Japan). The p53-responsive reporter plasmid (p53-Luc) and the 5x GAL4-binding element reporter plasmid (pFR-Luc) were purchased from Stratagene. The other p53 reporter plasmids, PG13-Luc and MG15-Luc, and the expression plasmids for the wild-type and mutant p53, pC53-WT and pC53-R273H, respectively, were provided by Dr Bert Vogelstein (Kern et al. 1992; El-Deiry et al. 1993). The pE2F-Luc (Azuma-Hara et al. 1999) and pcDNA3-mCBP (Miyagishi et al. 2000) plasmids were provided by Drs Taichi Uetsuki and Akiyoshi Fukamizu, respectively. The pSG5-16E6-AU1 plasmid was provided by Dr Richard Schlegel through Dr Peter Howley (Sherman & Schlegel 1996; Talis et al. 1998).
To construct expression plasmids for the C-terminally Flag-tagged human SEI proteins (pEF-SEI-Flag series), a DNA fragment encoding the Flag sequence (EFDYKDDDDK) was ligated to the 3' end of each coding sequence and inserted into the expression vector pEF-BOS-EX carrying the EF-1 enhancer/promoter (Murai et al. 1998). Expression plasmids for mouse SEI proteins were constructed similarly, but without the epitope tag. The ING expression plasmids, pEF-HA-ING1b and pEF-HA-ING2, were constructed by cloning each ING cDNA into the pEF-HA plasmid. The tetracycline (Tc)-responsive expression plasmids (pTRE-SEI-Flag series) were constructed by introducing DNA fragments encoding each Flag-tagged SEI protein into the pTRE2 vector (BD Clontech). An autoregulatory expression plasmid for reverse Tc-controlled transactivator (rtTA), pArtTApuro, was constructed by introducing the rtTA-encoding region of the pUHD172-1neo plasmid and an expression cassette for the Puro-resistance gene into the pUHD10-3 plasmid (provided by Dr Hermann Bujard) (Gossen et al. 1995). To construct an p53-shRNA expression plasmid, two oligonucleotides, 5'-caccGACTCCAGTGGTAATCTACttcaagagaGTAGATTACCACTGGAGTCttttt-3' and 5'-gcataaaaaGACTCCAGTGGTAATCTACtctcttgaaGTAGATTACCACTGGAGTC-3' (Brummelkamp et al. 2002), were annealed, and cloned into the BspMI-digested pcPUR+U6icassette vector (provided by Dr Kazunari Taira) (Miyagishi & Taira 2003) to yield the pPURU6-p53-1 plasmid. The Puro-resistance gene of this plasmid was replaced by a neomycin-resistance gene cassette to generate the pNEOU6-p53-1 plasmid.
Cell culture and establishment of stable transformants
HeLa cells and its derivatives were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 5% fetal calf serum (FCS, Invitrogen). All other cell lines were cultured in DMEM with 10% FCS. Transfection of cultured cells with plasmid DNA was carried out by the calcium phosphate co-precipitation method using Bes-buffered saline. HAM3, a HeLa-Tet-On cell line, was established by transfecting HeLa cells with the pArtTApuro plasmid. U2OS-Tet-On cells were purchased from BD Clontech. To establish clones that express SEI-Flag proteins in a Dox-inducible manner, HAM3 and U2OS-Tet-On cells were co-transfected with a hygromycin-resistance plasmid (pMiw-Hph) and the respective pTRE-SEI-Flag plasmid, and the resulting hygromycin B (Hyg)-resistant clones (0.5 mg/mL Hyg for HAM3 and 0.2 mg/mL Hyg for U2OS-Tet-On) were tested for the Dox-induced expression of each SEI-Flag protein by immunoblotting with the anti-Flag monoclonal antibody (mAb). To establish p53-knockdown cells, HeLa or HAM3-derived cells were transfected with the pNEOU6-p53-1 plasmid, and the resulting G418-resistant clones were isolated and tested for the reduction in p53 protein levels by immunoblotting using anti-p53 DO-1 mAb.
Luciferase reporter assay
Cells were seeded at 5 x 104 cells/well into a 24-well plate, cultured for 24 h, and transfected with 1 µg of a plasmid mixture, using the calcium phosphate co-precipitation method. Typically, 200 ng of reporter plasmid such as p53-Luc and 800 ng of single or combined effector plasmids were used. At 4 h after transfection, the transfected cells were washed with serum-free DMEM, fed with fresh medium containing FCS, and further cultured at 37 °C for 24 h. The cells were then washed and lyzed, and the luciferase activity was quantified as described (Fukumoto et al. 2001). The error bars in the figures indicate standard errors of the mean (SEM) from two independent transfection experiments.
Immunoprecipitation and immunoblot analyses
Cells grown in 60 mm-dishes were washed with ice-cold PBS and lyzed in 0.5 mL of NLB (NP-40 Lysis Buffer) as described (Fukunaga & Hunter 1997), and the supernatant was fractionated by SDS-polyacrylamide gel electrophoresis and transferred to a PVDF membrane filter (Immobilon-P, Millipore). Blotting was performed with mouse anti-Flag (M2, Sigma) mAb, anti-p53 mAb (DO-1, Santa Cruz), anti-p21 mAb (CP36-74, Upstate), or anti-mouse CBP polyclonal antibody (pAb) (CBP-CT, Upstate), together with horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG antibody (DAKO). Immunolabeled bands were detected using the enhanced chemiluminescence detection system (PerkinElmer). For immunoprecipitation of Flag-tagged SEIs, 15 µL of anti-FLAG M2 affinity gel (Sigma) was added to 0.3 mL of cell lysate, incubated for 2 h at 4 °C with gentle rotation, and washed five times with 1 mL of NLB. For the immunoblotting of the immunoprecipitated SEI-Flag, biotinylated anti-Flag mAb (M2, Sigma) was used together with HRP-streptavidin (Roche).
Real-time RT-PCR analysis
For the RT-PCR reaction, cDNA was synthesized from DNase I-treated total RNA (0.5 µg) using oligo-(dT) primer and Superscript III (Invitrogen) in a 10-µL reaction mixture. Real-time RT-PCR analysis was carried out using the LightCycler-FastStart DNA Master SYBR Green I kit (Roche). In brief, an aliquot of the synthesized cDNA was diluted with 20 µL of reaction mixture (3 mM MgCl2, 1 x FastStart DNA Master SYBR Green I) containing 10 pmol each of the sense and anti-sense primers, and the mixture was subjected to PCR in a LightCycler (Roche) under the following conditions: 40 cycles of 15 s at 95 °C, 5 s at 60 °C, and 20 s at 72 °C. The primer sets used were; 5'-GGCAGACAGCCTTCTGGCTA-3' and 5'-TCCAGTCCATCGTCCAGCAG-3' for mouse SEI-1, 5'-CCTCCCAGCAGAGAAGGACA-3' and 5'-TCAAGTCTGTCAGGAAACCC-3' for mouse SEI-2, 5'-AGGAGGAAATGAGCCAGGAT-3' and 5'-GGAGCTCGGAAACCGAGTTT-3' for mouse SEI-3, 5'-ACATTGAGGGCCTGAGTCAG-3' and 5'-TCAAGCCCATCGTCCAGTAG-3' for human SEI-1, 5'-CCTCTTGCCAGAAAAGGACA-3' and 5'-TCAAGTCTGTCAGGAAACCC-3' for human SEI-2, and 5'-CTGGTGAAGTTGCAGCTTTG-3' and 5'-GGCAAAGGTCAGAAACTGGA-3' for human SEI-3. In all real-time RT-PCR experiments, cloned cDNA fragments (102–106 copies per reaction) containing the respective target region were amplified in parallel, and used as standards. Data from real-time RT-PCRs were normalized against the standards and expressed as the copy number of the target mRNA per ng of total RNA.
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Acknowledgements |
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Footnotes |
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* Correspondence: E-mail: fukunaga{at}genetic.med.osaka-u.ac.jp
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Received: 9 April 2005
Accepted: 16 May 2005
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 Y. Isegawa, Y. Miyamoto, Y. Yasuda, K. Semi, K. Tsujimura, R. Fukunaga, A. Ohshima, Y. Horiguchi, Y. Yoneda, and N. Sugimoto Characterization of the Human Herpesvirus 6 U69 Gene Product and Identification of Its Nuclear Localization Signal J. Virol., January 15, 2008; 82(2): 710 - 718. [Abstract] [Full Text] [PDF]
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R. Hayashi, Y. Goto, R. Ikeda, K. K. Yokoyama, and K. Yoshida CDCA4 Is an E2F Transcription Factor Family-induced Nuclear Factor That Regulates E2F-dependent Transcriptional Activation and Cell Proliferation J. Biol. Chem., November 24, 2006; 281(47): 35633 - 35648. [Abstract] [Full Text] [PDF]
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) with 1 µg/mL Dox or left untreated (
, 
) (day 0). Every day, the cells in two wells were trypsinized and the cell number in each 1/20 portion of the cell suspensions was counted. The average number from the two wells is plotted. To maintain the SEI protein expression, the culture medium was replaced with fresh medium containing 1 µg/mL Dox every other day. (C) Representative p53-knockdown clones, PG8, KG10, and SG6, derived from H4.8 (SEI-1), H3.4 (SEI-2), and H2.4 (SEI-3), respectively, were Dox-treated or left untreated for 24 h, and the cell lysates were analyzed by immunoblotting using the anti-p53 mAb (DO-1). Neo-resistant clones that were isolated by the transfection of the respective parental cells with the empty vector were analyzed in parallel as vector control clones (VC). (D) For each SEI-expressing parental clone, one control (VC, •, 
, 
) were treated with Dox (•,