Published Ahead of Print 11 June 2007. 10.1128/MCB.00603-07.
2007, 27(16):5898. DOI:Mol. Cell. Biol. Liuh-Yow Chen, Dan Liu and Zhou Songyang
Control of Telomeric Proteins
Telomere Maintenance through Spatial mcb.asm/content/27/16/5898Updated information and rvices can be found at: The include:
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M OLECULAR AND C ELLULAR B IOLOGY,Aug.2007,p.5898–5909Vol.27,No.16 0270-7306/07/$08.00ϩ0doi:10.1128/MCB.00603-07
Copyright©2007,American Society for Microbiology.All Rights Rerved.
重庆心理咨询师培训
Telomere Maintenance through Spatial Control of Telomeric Proteinsᰔ
四六级准考证查询系统
Liuh-Yow Chen,Dan Liu,and Zhou Songyang*
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Verna and Marrs McLean Department of Biochemistry and Molecular Biology,Baylor College of Medicine,
One Baylor Plaza,Houston,Texas77030
Received4April2007/Returned for modification1May2007/Accepted27May2007
The six human telomeric proteins TRF1,TRF2,RAP1,TIN2,POT1,and TPP1can form a complex called
the telosome/shelterin,which is required for telomere protection and length control.TPP1has been shown
to regulate both POT1telomere localization and telosome asmbly through its binding to TIN2.It
remains to be determined where such interactions take place and whether cellular compartmentalization
of telomeric proteins is important for telomere maintenance.We systematically investigated here the
cellular localization and interactions of human telomeric proteins.Interestingly,we found TIN2,TPP1,
and POT1to localize and interact with each other in both the cytoplasm and the nucleus.Unexpectedly,
TPP1contains a functional nuclear export signal that directly controls the amount of TPP1and POT1in
the nucleus.Furthermore,binding of TIN2to TPP1promotes the nuclear localization of TPP1and POT1.
We also found that disrupting TPP1nuclear export could result in telomeric DNA damage respon and
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telomere length disregulation.Ourfindings highlight how the coordinated interactions between TIN2,
TPP1,and POT1in the cytoplasm regulate the asmbly and function of the telosome in the nucleus and
indicate for thefirst time the importance of nuclear export and spatial control of telomeric proteins in
telomere maintenance.
Telomere maintenance by telomera and telomeric proteins is esntial for normal cell growth,and its disregulation may have direct conquences in aging and cancer(2,10,11,22,28,29,44). Recent biochemical and genetic experiments have led to a much clearer understanding of how telomere ends are protected in mammalian cells.To date,six major telomeric proteins—namely, TRF1,TRF2,RAP1,TIN2,POT1,and TPP1(previously PTOP, PIP1,or TINT1)—have been identified in human cells and shown to participate in telomere regulation(2,9,38).The proteins perform related but distinct functions at the telomeres.Under-standing the function and molecular interaction of the telo-meric proteins is fundamental to telomere biology. Telomeres in mammalian cells consist of long telomeric dou-ble-stranded DNA(dsDNA)repeats and short single-stranded DNA(ssDNA)overhangs(12,13).TRF1and TRF2directly associate with the repeats through their myb domains(4). TRF1is a negative regulator of telomere length since overex-pression of TRF1results in gradual shortening of telomeres (41).Overexpression of dominant-negative mutants or dele-tion of TRF2results in DNA damage respons at the telo-meres and chromosomal end-to-end fusions,underlining its esntial role in telomere end protection(5,42).TRF2also works cloly with its
associated protein RAP1(24,31).In TRF2KO mou embryonicfibroblast cells,protein levels of RAP1,but not TRF1,were diminished,suggesting that TRF2 and RAP1act together(5).
The human telomere ssDNA overhangs are protected by POT1and TPP1.POT1and TPP1are OB-fold-containing proteins that are structurally homologous to ciliate telomere end binding proteins TEBP-␣and-(16,43,46).Originally identified in yeast,POT1family proteins have been shown to regulate telomere length and protect telomeres from DNA damage,rapid degradation,and chromosomal fusion(6,7,14, 19,27,36,45).Human POT1harbors two OB folds in its N terminus and recognizes telomeric ssDNA with high affinity (23).Surprisingly,the telomeric targeting of POT1is con-trolled by TPP1,a POT1-and TIN2-interacting protein(26,46, 48).However,whether TPP1controls POT1telomere target-ing by regulating POT1nuclear localization remains to be determined.TPP1heterodimerizes with POT1and enhances its affinity toward telomere ssDNA(43,46).In addition to binding and protecting the G-strand telomere overhangs,the POT1-TPP1complex is also capable of recruiting and stimu-lating telomera activity,thereby regulating telomere length through TPP1-telomera interaction(43,46).Elucidating the interaction and the function of the two proteins should con-tinue to prove pivotal to our understanding of telomere main-tenance.
Human telomeres are regulated by various telomeric protein complexes,including the high-molecular-weight telosome/shel-terin that contains all six major telomeric proteins(9,25). Within the telosome,TRF1and TRF2are connected to POT1 through TIN2and TPP1,two key factors for telosome asm-bly(32).In the abnce of TIN2,only smaller telomeric sub-complexes were formed(32).TIN2has emerged as the key telosome element becau of its ability to interact with TRF1, TRF2,and TPP1(17,21,25,26,47).Meanwhile,TPP1pro-motes the TRF1-TIN2-TRF2subcomplex and telosome for-mation through TPP1-TIN2interaction(32).Such interdepen-dence during high-order complex formation argues that the spatial and/or temporal restriction of telomeric protein-protein interactions may determine the ultimate composition and func-tion of telomeric protein complexes.Although there have been numerous studies on the function of telomeric proteins in the
*Corresponding author.Mailing address:Verna and Marrs McLean Department of Biochemistry and Molecular Biology,Baylor College of Medicine,One Baylor Plaza,Houston,TX77030.Phone:(713)798-5220.Fax:(713)796-9438.E-mail:du.
ᰔPublished ahead of print on11June2007.
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nucleus,it remains unclear,for example,whether the forma-tion of various telomeric complexes can be directly regulated by the subcellular localization of telomeric proteins.
Apart from their known telomeric association,little is known regarding the total cellular distribution of the six mammalian telomeric proteins.To visualize telomere targeting of proteins by immunostaining,cells are often doubly permeabilized(24). This method disrupts proteins in both the cytoplasm and the nucleoplasm,providing a much more limited view of localiza-tion of telomeric proteins inside the cell.In order to under-stand the role of cellular localization of telomeric proteins in telomere maintenance,we have systematically investigated the localization and in vivo interactions of the six human telomeric proteins.Notably,we found TIN2,TPP1,and POT1to localize and interact with each other not only in the nucleus but also in the cytoplasm.Unexpectedly,we found TPP1to contain a nuclear export signal.We showed here that TPP1shuttles between the cytoplasm and the nucleus,which in turn regulates the nuclear localization of POT1.In addition,TIN2binding to TPP1helps to promote the nuclear retention of POT1.Fur-thermore,disruption of TPP1nuclear export results in telo-mere dysfunction.Our results suggest that the coordinated interactions between TIN2,
TPP1,and POT1in the cytoplasm regulate the asmbly and function of the telosome in the nucleus,and underscore the importance of spatial control of telomeric proteins and their interactions in regulating POT1 localization and telomere maintenance.
MATERIALS AND METHODS
Expression constructs,cell cultures,and antibodies.Rabbit anti-TPP1and anti-TIN2(26),anti-RAP1(Bethyl),and goat anti-TRF1(32),anti-POT1(NB500-176;Novus Biologicals),anti-TRF2(OP129;Calbiochem),anti-GRB2(catalog no. 610112;BD Transduction Laboratories),anti-␣-tubulin(T9026;Sigma),anti-lamin (sc-6215;Santa Cruz),anti-ORC2(catalog no.551179;BD Transduction Laborato-ries),and anti-Flag(M2;Sigma)antibodies were ud for Western blot analysis. Anti-POT1N and anti-53BP1(46),anti-TRF2(OP129;Calbiochem),and anti-Flag (M2;Sigma)antibodies were ud for immunostaining.
To generate cell lines stably expressingfluorescence protein-tagged or Flag-tagged telomeric proteins,Gateway cloning(Invitrogen)was performed to clone the cDNAs into pCL-bad Gateway destination vectors containing greenfluo-rescent protein(GFP),yellowfluorescent protein(YFP),cyanfluorescent pro-tein(CFP),DsRed,or Flag.All of thefluorescence proteins were fu
d to the N terminus of the targeting proteins.V5-tagged POT1was generated by TOPO cloning into pDNA3-bad vector(Invitrogen).Mutations on TPP1were gener-ated by QuikChange mutagenesis(Stratagene).To generate cell lines stably expressing telomeric proteins fud to the N-or C-terminal fragments of YFP, the cDNAs of the telomeric proteins were Gateway cloned into pBabe-bad retroviral vectors containing the quences encoding either YFPn(amino acids 1to155of Venus YFP)or YFPc(amino acids156to239of YFP).To generate cell lines stably expressing short hairpin RNAs(shRNAs)specifically targeting TPP1or GFP,mou U6promoter-driven expression of shRNA constructs were generated on a pCL-bad vector.The RNA interference quences for sh-TPP1-A and shGFP are5Ј-GTGGTACCAGCATCAGCCTT-3Јand5Ј-CACA AGCTGGAGTACAACT-3Ј(32),respectively.
kg是什么意思Stable cells expressing various constructs were established by retroviral infec-tion.The cells were lected in the prence of puromycin,G418,or both.The lected cells were maintained as a pool for experiments described here. Coimmunoprecipitation.To prepare cell lysates for coimmunoprecipitation, the cells were lyd in NETN(1M Tris[pH8.0],1mM EDTA,100mM NaCl, 0.5%NP-40)buffer supplemented with protea inhibitor cocktails(Sigma)and subjected to centrifuge at14,000ϫg for15min.After centrifugation,the supernatant was ud for immunoprecipitation with anti-
Flag M2affinity resins (Sigma)on ice for1h.After four washes with NETN buffer,the proteins were eluted with Flag peptide(200g/ml)and subjected to Western blot analysis. Immunofluorescence and BiFC.Cells for immunofluorescence were grown on glass coverslips.The cells were thenfixed with4%paraformaldehyde prepared in phosphate-buffered saline for15min and permeabilized with0.2%Triton X-100.After blocking with1%goat rum,the proteins were detected with appropriate antibodies,followed by condary antibodies conjugated withfluo-rescein isothiocyanate or Texas red.To detect telomere dysfunction-induced foci, the cells were costained with anti-TRF2monoclonal and anti-53BP1polyclonal antibodies.To visualize the nuclei,DAPI(4Ј,6Ј-diamidino-2-phenylindole)was ud to stain DNA.Fluorescence microscopy was performed on a Nikon TE200 microscope equipped with a Coolsnap-fx charge-coupled device camera.GFP fusion proteins are ready for analysis afterfixation.For bimolecularfluorescence complementation(BiFC)experiments,the cells were grown on glass-bottom culture plates(MatTek Corp.).The cell nuclei were stained with Hoechst33342, a cell-permeable DNA dye.Thefluorescence emissions of the living cells were imaged as described above.
Subcellular fractionation.A total of106of HTC75cells were treated with trypsin,washed with culture medium and PBS,and resuspended in20l of hypotonic buffer A(10mM HEPES[pH7.9],10mM KCl,1.
5mM MgCl2,0.34 M sucro,10%glycerol,1mM dithiothreitol,and protea inhibitors).A lim-iting detergent lysis was performed bythe addition of0.1%of Triton X-100to the cells,followed by incubation at4°C for5min.After incubation,the lysates were centrifuged at1,300ϫg for4min.After centrifugation,the supernatant and the pellet were saved as the cytosolic and the nuclear/heavy membrane fractions, respectively,for Western blot analysis.
In vitro pull-down assay.The glutathione S-transfera(GST)–POT1protein was produced in Sf9inct cells and purified by glutathione agaro as previously described(46).The various TPP1proteins labeled with[35S]methionine were prepared by using TnT quick-coupled transcription/translation systems(Pro-mega).The binding reaction of GST-POT1with TPP1proteins was performed in NETN buffer at4°C for1h.The precipitates were washed with NETN buffer, eluted with sodium dodecyl sulfate(SDS)sample buffer,and resolved by SDS-polyacrylamide gel electrophoresis(PAGE).
Telomere restriction fragment assay.HTC75cells stably expressing various TPP1proteins were generated by retrovirus infections and subquent puromy-cin lection.After drug lection(at which point designated as PD0),the surviving cells were passaged and collected at various time points.The genomic DNA was extracted with a DNeasy kit(QIAGEN)and ud for telomere re-striction fragment
assay performed as previously described(26).The data were then analyzed by using ImageQuant and the Telorun analysis tool(33).
RESULTS
Intracellular localization of telomeric proteins.Numerous studies have established the critical roles for the six telomeric proteins in regulating mammalian telomere chromatin(1,2,9, 34,38).The majority of the studies have focud on the telomere functions of the proteins;the cellular localization of the proteins remains poorly characterized.We therefore t out to systematically investigate the localization of the six telomeric proteins in human cells.To study the localization of human telomeric proteins,we generated cell lines stably ex-pressing GFP-tagged TRF1,TRF2,RAP1,TPP1,TIN2,and POT1and examined their localization.Human TPP1mRNA contains two potential Kozak quences for translation initia-tion,which encodes,respectively,the long-form TPP1-L(544 amino acids)and the short-form TPP1-S(458amino acids,also called TPP1⌬86).The additional N-terminal86amino acids in TPP1-L appear to be abnt in other species,including the mou(26).Mass spectrometry quencing and Western blot-ting experiments suggest that TPP1-S is the predominant form of TPP1in human cells(data not shown).We therefore fo-cud on TPP1-S for the prent study.
Interestingly,the GFP-tagged telomeric proteins exhibited dif-ferent localization patterns.While GFP-TRF1,-TRF2,-RAP1, and-TIN2localized primarily in the nucleus(Fig.1A),GFP-TPP1and GFP-POT1were clearly prent in both the nucleus and the cytoplasm(Fig.1A).A small amount of GFP-TIN2could
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yue什么意思also be detected in the cytoplasm.On clo examination,all GFP fusion proteins showed telomere localization (data not shown).To further confirm this obrvation,cytoplasmic and nuclear fractions of HTC75cells were prepared by limiting detergent lysis for Western blotting (30).As shown in Fig.1B,marker proteins for the S1cytoplasmic fraction (GRB2and tubulin)and the P1nuclear/heavy membrane fraction (lamin A/C and ORC2)were largely consistent with their respective localiza-tions.A trace amount of cytoplasmic marker proteins in the nuclear fraction was likely a result of incomplete cell lysis.In agreement with previous findings,all six telomere proteins mainly localized to the nucleus.In particular,endogenous TIN2,TRF2,TRF1,and RAP1are primarily nuclear localized,a fi
nding consistent with published studies (24,37).Impor-tantly,a significant amount of endogenous TPP1and POT1was also detected in the cytoplasmic faction (Fig.1B).Taken together,the data are consistent with our GFP fusion protein analys (Fig.1A)and demonstrate that TPP1and POT1may also localize to the cytoplasm.
TIN2,TPP1,and POT1interact with each other in both the cytoplasm and the nucleus.Given their critical roles in regu-lating telomere length and protecting telomere ends,the nu-clear asmbly and telomere targeting of the six main telomere proteins must be esntial for telomere maintenance.How-ever,the cytoplasmic localization of TPP1and POT1suggests that the three proteins may also interact with each other in the cytoplasm.In order to verify where interactions of telo-meric proteins occur in live cells,we utilized BiFC to analyze pairwi interactions among TRF2,TIN2,TPP1,and POT1(18).Previously,we and other groups demonstrated that TIN2binds directly to TRF2and TPP1,whereas TPP1interacts directly with POT1(Fig.2A)(17,21,25,26,48).
In BiFC assays,two proteins are fud to parate fragments of YFP.When the proteins interact,the YFP fragments will be brought into clo proximity to form a functional fluores-cent complex (Fig.2B).As expected,YFP signals could be detected in cells coexpressing TIN2-TRF2,TPP1-TIN2,and TPP1-POT1pairs,but not the YFP fragments alone (Fig.2C).The TIN2-TRF2int
eraction appeared to occur largely (Ͼ95%)in the nucleus.Furthermore,the signals in the cells displayed the punctate pattern that was superimposable with the telomere marker dsRed-TRF1.In contrast,in all of the fluorescence-positive interpha cells examined,TIN2-TPP1and POT1-TPP1interactions took place in both the cytoplasm and the nucleus (Fig.2C).In the nucleus,the punctate YFP fluorescence signals overlapped with dsRed-TRF1,
indicating
FIG.1.Subcellular localization of human telomeric proteins.(A)Localization of GFP-tagged human telomeric proteins.HTC75cells were infected with retroviral vectors for stable expression of different GFP-tagged telomeric proteins.The cells were fixed,and fluorescence signals were visualized by using a microscope.The cell nuclei were stained with DAPI.(B)Fractionation and Western blot analysis of endogenous telomeric proteins.HTC75cells were fractionated into cytoplasmic (S1)and nuclear/heavy membrane (P1)fractions as described in Materials and Methods and resolved by SDS-PAGE.Whole-cell extracts (WCE)were also prepared.Western blotting was performed with antibodies for cytoplasmic marker proteins (GRB2and tubulin),nuclear marker proteins (lamin A/C and ORC2),and telomeric proteins (TPP1,POT1,TIN2,TRF1,TRF2,and RAP1).An arrow indicates endogenous TPP1or POT1.POT1has a 70-kDa and a 55-kDa form (15).
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briskly
that TIN2-TPP1and POT1-TPP1interactions could occur at the telomeres (Fig.2C).In the cytoplasm,the fluorescence signals appeared evenly distributed.As a negative control,cells that coexpresd TIN2with the TPP1mutant (TPP1⌬C22)that no longer binds TIN2(32)failed to exhibit fluorescence in the BiFC assay (Fig.2D),suggesting that fluorescence comple-
mentation required direct interactions between TPP1and TIN2.The data are consistent with our obrved cytoplasmic localization of TPP1and POT1(Fig.1)and rai the question of whether the interactions of telomeric proteins and the likely formation of subcomplexes in different subcellular compart-ments may affect the function of the proteins at the telo-meres.
Regulation of POT1and TPP1cellular localization by TIN2.Previous work has demonstrated that TIN2,TPP1,and POT1interact with each other in the order of TIN2-TPP1-POT1to form the telosome (32).TPP1controls POT1telomere local-ization,which is in turn regulated by TIN2-TPP1interaction.Given the data in Fig.1and 2,we next sought to determine whether TIN2could modulate TPP1and POT1cellular local-ization.We reasoned that TIN2might regulate TPP1and POT1telomere localization through the nuclear import and/or retention of TPP1or POT1.The higher intensity of GFP-TPP1and GFP-POT1in the cytoplasm may be due to overexpression of the proteins and limited endogenous TIN2.If this is the ca,overexpression of TIN2in GFP-TPP1or GFP-
POT1cells should lead to incread GFP-TPP1and GFP-POT1in the nucleus.As shown in Fig.3A,in all of the interpha cells examined (ϳ100%),GFP-TPP1was predominantly found in the cytoplasm.However,coexpression of dsRed-tagged TIN2resulted in incread nuclear TPP1signal in all of the inter-pha cells examined,suggesting that TIN2-TPP1interaction promotes nuclear localization of TPP1.In further support of this notion,the TPP1mutant (TPP1⌬C22)that no longer binds TIN2remained cytoplasm localized when TIN2was coex-presd (Fig.3A).
Similar to TPP1,POT1nuclear localization appeared to be regulated by TIN2as well.As shown in Fig.3B and E,coex-pression of CFP-TIN2concentrated POT1in the nucleus.Be-cau of the TIN2-TPP1-POT1link,we speculated that TIN2might increa POT1nuclear retention through TPP1.If this is the ca,reducing endogenous TPP1level by RNA interfer-ence should affect nuclear localization of POT1.Indeed,in TPP1knockdown cells,coexpression of TIN2failed to pro-mote YFP-POT1accumulation in the nucleus (Fig.3C,D,and E).The results are not only consistent with our model that the TIN2-TPP1-POT1link is esntial for POT1nuclear local-ization but also suggest that POT1activity is controlled by TIN2and TPP1through both nuclear localization and telo-mere targeting.
TPP1shuttles between the cytoplasm and the nucleus.TPP1is a key factor in telosome asmbly an
d regulates POT1lo-calization (26,32).The finding of TPP1residing in both the cytoplasm and the nucleus prompted us to investigate the mechanism that controls TPP1subcellular localization.We hypothesized that TPP1localization might be regulated by nuclear import or export.To investigate this possibility,cells stably expressing GFP-TPP1were treated with leptomycin B (LMB).LMB inhibits CRM-1/exportin 1,a protein necessary for nuclear export signal (NES)-mediated protein export.As shown in Fig.4A,the accumulation of wild-type TPP1in the nucleus was found in LMB-treated cells,suggesting that TPP1can be exported to the cytoplasm in a CRM-1-dependent man-ner.When the LMB-treated cells were further incubated in the abnce of LMB,translocation of TPP1to the cytoplasm could be obrved within 1h.The results suggest that
辞典
TPP1
FIG.2.In vivo interactions among human telomeric proteins revealed by BiFC.(A)Diagram of known pairwi interactions within the telo-some.(B)Schematic reprentation of the BiFC technique in detecting TIN2-TRF2interaction.(C)Visualization of telomeric protein interac-tions in live cells through BiFC.HTC75cells stably coexpressing YFP fragments alone or YFP fragment-tagged TRF2,TIN2,TPP1,or POT1(as indicated)were established.YFPn,Venus YFP N-terminal fragment (amino acids 1to 155);YFPc,YFP C-terminal fragment (amino acids 156to 239).To detect telomeres in the cells,dsRed-TRF1was also coex-presd.The cells were stained with Hoechst 33342to visualize the nuclei and analyzed directly under a fluorescence microscope.(D)Positive BiFC fluorescence signal by YFPn-TPP1and YFPc-TIN2required direct inter-action between TPP1and TIN2.HTC75cells coexpressing YFP frag-ments alone or YFP fragment-tagged TIN2,wild-type,or mutant TPP1were analyzed by fluorescence-activated cell sorting.The percentages of GFP-positive cells were indicated in the histograms.Control,parental HTC75cells.
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is imported into the nucleus and then exported back to the cytoplasm.
To identify the domain that is responsible for TPP1nu-clear import or export,we examined the localization of TPP1deletion mutants.Deletion of the POT1recruitment domain (TPP1⌬RD)enabled TPP1to accumulate in the nucleus in ϳ100%of the interpha cells (Fig.4B),suggest-ing that cytoplasmic localization of TPP1may be maintained by either TPP1interaction with POT1or an active NES located within the RD domain.Interestingly,disruption of the TIN2-interacting domain (TPP1⌬C22)did not change TPP1localization.In addition,further mutation of the TIN2binding site on TPP1⌬RD did not prevent its nuclear reten-tion (Fig.4B),implying independent mechanisms for nu-clear export mediated through the RD domain versus nu-clear retention mediated through TIN2-TPP1
interaction.
FIG.3.TIN2promotes TPP1and POT1nuclear localization.(A)TIN2increas nuclear TPP1through TIN2-TPP1interaction.Cells stably expressing GFP-TPP1or GFP-TPP1⌬C22alone or with dsRed-TIN2were fixed,and the localization of GFP-TPP1proteins was determined by fluorescence microscopy.(B)TIN2increas nuclear POT1.Cells stably expressing YFP-POT1with or without CFP-TIN2were fixed,and the localization of YFP-POT1proteins was determined by fluorescence microscopy.(C)Knockdown of TPP1in HTC75cells.Stable TPP1knockdown cells were established by transduction with retroviral vectors expressing a control shRNA quence against GFP (shGFP)or a shRNA quence (shTPP1-A)against human TPP1.Whole-cell extracts from the cells were Western blotted for endogenous TPP1(marked by the arrow),TRF2,RAP1,and tubulin.Tubulin was ud as a loading control.(D)TIN2promotes POT1nuclear retention in a TPP1-dependent manner.Cells stably expressing YFP-POT1with or without Flag-TIN2and cells expressing YFP-POT1with Flag-TIN2and TPP1shRNA-A were generated.The cells were fixed,and the localization of YFP-POT1proteins was determined by fluorescence microscopy.Nuclei were detected with DAPI staining.(E)The data in panel D were quantified bad on the percentages of cells with cytoplasmic and nuclear YFP-POT1.
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