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Clearance of p16Ink4a-positive senescent cells delays

LETTER

doi:10.1038/nature10600 Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders

Darren J.Baker1,2,3,Tobias Wijshake1,4,Tamar Tchkonia3,Nathan K.LeBrasseur3,5,Bennett G.Childs1,Bart van de Sluis4, James L.Kirkland3&Jan M.van Deursen1,2,3

Advanced age is the main risk factor for most chronic diseases and

functional deficits in humans,but the fundamental mechanisms that drive ageing remain largely unknown,impeding the develop-

ment of interventions that might delay or prevent age-related dis-

orders and maximize healthy lifespan.Cellular senescence,which halts the proliferation of damaged or dysfunctional cells,is an

important mechanism to constrain the malignant progression of

tumour cells1,2.Senescent cells accumulate in various tissues and organs with ageing3and have been hypothesized to disrupt tissue

structure and function because of the components they secrete4,5.

However,whether senescent cells are causally implicated in age-related dysfunction and whether their removal is beneficial has

remained unknown.To address these fundamental questions,we

made use of a biomarker for senescence,p16Ink4a,to design a novel transgene,INK-ATTAC,for inducible elimination of p16Ink4a-positive senescent cells upon administration of a drug.Here we

show that in the BubR1progeroid mouse background,INK-ATTAC removes p16Ink4a-positive senescent cells upon drug treat-ment.In tissues—such as adipose tissue,skeletal muscle and eye—

in which p16Ink4a contributes to the acquisition of age-related pathologies,life-long removal of p16Ink4a-expressing cells delayed

onset of these phenotypes.Furthermore,late-life clearance attenuated

progression of already established age-related disorders.These data

indicate that cellular senescence is causally implicated in generat-ing age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.

To examine the role of cellular senescence in ageing and age-related pathologies,we designed a transgenic strategy for the clearance of senescent cells in mice.We based our approach on an earlier mouse model,termed FAT-ATTAC(fat apoptosis through targeted activation of caspase),in which adipocytes were selectively killed by apoptosis upon the administration of AP20187,a synthetic drug that induces dimerization of a membrane-bound myristoylated FK506-binding-protein–caspase8(FKBP–Casp8)fusion protein expressed specifically in adipocytes via the minimal Fabp4promoter6.Although a universal marker that is solely expressed in senescent cells has not been identified, most senescent cells seem to express p16Ink4a,a cyclin-dependent kinase inhibitor and tumour suppressor that enforces growth arrest by activ-ating Rb5,7.Additionally,the expression of p16Ink4a is known to increase with ageing in several rodent and human tissues8.We replaced the Fabp4promoter with a2,617-bp fragment of the p16Ink4a gene pro-moter that is transcriptionally active in senescent,but not non-senescent cells(Fig.1a)9.We added an internal ribosome entry site (IRES)followed by an open reading frame(ORF)coding for enhanced green fluorescence protein(EGFP)to allow for detection and collection of p16Ink4a-positive senescent cells.Injection of the resulting construct into fertilized eggs yielded nine transgenic INK-ATTAC founder lines. To examine whether removal of p16Ink4a-expressing cells is tech-nically feasible and whether this affects age-associated deficits in mice,we bred each of the founder lines onto a BubR1hypomorphic (BubR1H/H)genetic background.BubR1encodes a key member of the mitotic checkpoint,a surveillance mechanism that ensures accurate chromosome segregation in mitosis by inhibiting the ubiquitin ligase activity of Cdc20-activated anaphase-promoting complex(APC Cdc20)in the presence of unattached chromosomes10,11.BubR1H/H mice have a markedly shortened lifespan and exhibit a variety of age-related pheno-types,including infertility,lordokyphosis,sarcopenia,cataracts,fat loss, cardiac arrhythmias,arterial wall stiffening,impaired wound healing and dermal thinning12–14.It has been proposed that BubR1is a deter-minant of natural ageing,because levels of BubR1decline markedly with age12–14.BubR1H/H mice selectively accumulate p16Ink4a-positive cells in certain tissues in which age-associated pathologies develop,including adipose tissue,skeletal muscle and eye15.Inactivation of p16Ink4a in these mice is known to delay the onset of age-related phenotypes selectively in these tissues15.To screen for INK-ATTAC transgene activity in p16Ink4a-positive cells,we collected samples of inguinal adipose tissue(IAT)from each of the nine BubR1H/H;INK-ATTAC strains at5months of age and analysed them for GFP expression by fluorescence microscopy.We observed GFP fluorescence in two of these strains,BubR1H/H;INK-ATTAC-3and-5(Fig.1b and Supplementary Fig.1a).Quantitative reverse transcription–polymerase chain reaction(qRT–PCR)analysis of various tissues from BubR1H/H;INK-ATTAC-3and-5mice demon-strated that INK-ATTAC and GFP transcript levels were significantly elevated in adipose tissue,skeletal muscle and eye,but not in tissues in which endogenous p16Ink4a is not induced,including liver and heart (Fig.1c and Supplementary Fig.1b).

To confirm that transgenic INK-ATTAC and endogenous p16Ink4a are under the same transcriptional control mechanism outside the context of BubR1hypomorphism,we harvested bone marrow cells from2-month-old wild-type(WT);INK-ATTAC-3and-5mice and cultured them in the absence or presence of rosiglitazone,a drug that can induce cellular senescence and p16Ink4a expression through activa-tion of PPAR c16.Immunofluorescence microscopy revealed that a high proportion of cells expressed Flag-tagged FKBP–Casp8in the presence of rosiglitazone,but not in its absence(Fig.1d).Furthermore,we observed selective INK-ATTAC transgene induction in tissues of WT;INK-ATTAC-3mice showing elevated expression of endogenous p16Ink4a upon chronological ageing(Supplementary Fig.2).Together, these data indicate that INK-ATTAC gene activity in founder lines3 and5overlaps with endogenous p16Ink4a expression.

Next,we tested whether INK-ATTAC is expressed in senescent cells in BubR1hypomorphic tissue.Fat tissue of aged BubR1H/H;INK-ATTAC mice stained strongly for senescence-associated-b-galactosidase (SA-b-Gal;Fig.1e).qRT–PCR analysis demonstrated that INK-ATTAC expression correlates with expression of senescence markers in IAT (Fig.1f and Supplementary Fig.3a).Skeletal muscle and lens tissue of aged BubR1H/H;INK-ATTAC mice are SA-b-Gal negative(data not shown),but both these tissues expressed other markers of senescence

1Department of Pediatric and Adolescent Medicine,Mayo Clinic College of Medicine,Rochester,Minnesota55905,USA.2Molecular Biology and Biochemistry,Mayo Clinic College of Medicine,Rochester, Minnesota55905,USA.3Robert and Arlene Kogod Center on Aging,Mayo Clinic College of Medicine,Rochester,Minnesota55905,USA.4Department of Pathology and Medical Biology,University Medical Center Groningen,Groningen University,Groningen9700RB,The Netherlands.5Physical Medicine and Rehabilitation,Mayo Clinic College of Medicine,Rochester,Minnesota55905,USA.

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(Fig.1f and Supplementary Fig.3a).Senescence markers were not elevated in 3-week-old BubR1H/H ;INK-ATTAC mice (Supplementary Fig.3b,c).To obtain additional evidence for selective expression of INK-ATTAC in senescent cells,we collected IAT from aged BubR1H/H ;INK-ATTAC animals,prepared single-cell suspensions by collagenase treatment,separated GFP 1and GFP –cell populations by fluorescence activated cell sorting (FACS;Fig.1g),and analysed each population for expression of INK-ATTAC and senescence markers by qRT–PCR.GFP 1cells not only expressed much higher levels of p16Ink4a than GFP –cells but also had elevated levels of other key senescence markers (Fig.1h and Supplementary Fig.3d).Furthermore,two conditions that induce p16Ink4a expression and senescence in primary mouse embryonic fibroblasts (MEFs),ectopic expression of oncogenic Ras and serial passaging 12,17,18,produced a subpopulation of

GFP 1WT;INK-ATTAC-3MEFs that,in contrast to the remaining GFP –cells,stained positively for SA-b -Gal (Fig.1i).Taken together,these results indicate that INK-ATTAC is selectively expressed in p16Ink4a -positive senescent cells.

To determine whether INK-ATTAC can eliminate senescent cells,we cultured bone marrow cells of WT;INK-ATTAC transgenic lines 3and 5in the presence of rosiglitazone to induce senescence and then monitored cell survival after activating the FKBP–Casp8fusion protein by AP20187treatment.We found that the vast majority of cells from both transgenic lines were either dead or in the process of dying 48h after adding AP20187(Fig.2a).In contrast,parallel cultures that remained untreated consisted almost entirely of viable SA-b -Gal-positive cells.These data show that FKBP–Casp8activation efficiently eliminates p16Ink4a -positive senescent cells in vitro .

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p 21 P a i 1 p 19 p 16 I l 6 Figure 1|Generation and characterization of INK-ATTAC transgenic

mice.a ,Schematic of the INK-ATTAC construct and the mechanism of apoptosis activation.b ,GFP intensity of IAT.c ,qRT–PCR analysis of the indicated tissues of 10-month-old mice.ATTAC,INK-ATTAC ;H/H ,BubR1H/H ;SkM,skeletal muscle (gastrocnemius).d ,Bone marrow cells harvested from 2-month-old mice immunostained for Flag after culture in the absence or presence of rosiglitazone for 48h.e ,SA-b -Gal stained IAT collected from 9-month-old mice of the indicated genotypes.f ,Expression of senescence markers in tissues of 10-month-old mice measured by qRT–PCR.

All increases are statistically significant (P ,0.05).g ,FACS profile of single-cell suspensions from IAT of 10-month-old mice.Brackets indicate sorting gates.h ,GFP 1and GFP –cell populations from IAT analysed for relative expression of senescence markers by qRT–PCR.All increases are statistically significant (P ,0.01).i ,Bright field images of MEFs sorted into GFP 1and GFP –

populations after induction of senescence and then stained for SA-b -Gal.For all experiments,n 53untreated females per genotype.Error bars,s.d.Scale bars in b ,d and i ,20m m.*P ,0.05,**P ,0.01,***P ,0.001.

RESEARCH LETTER

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Next,we examined whether clearance of p16Ink4a

-expressing cells

from BubR1H/H

mice prevents or delays the onset of age-related phenotypes in this progeroid background.To this end,we established cohorts of BubR1H/H ;INK-ATTAC-3and -5mice,which were either treated with AP20187every third day beginning at 3weeks of age or left untreated.Both treated and untreated mice were monitored for the development of age-associated deficits known to accompany p16Ink4a induction,including sarcopenia,cataracts and loss of adipose tissue 15.Remarkably,treated mice of both BubR1H/H ;INK-ATTAC lines had substantially delayed onset of lordokyphosis (a measure of sarcopenia onset in this model 15)and cataracts compared to untreated mice (Fig.2b,c).Consistent with decreased lordokyphosis,muscle fibre diameters of AP20187-treated BubR1H/H ;INK-ATTAC animals were larger than those of untreated counterparts (Fig.2d).In addition to muscle retention,treadmill exercise tests revealed that duration of exercise,distance travelled and overall amount of work performed were all significantly increased in the animals treated with AP20187(Fig.2e),indicating preservation of muscle function.Dual-energy X-ray absorptiometry (DEXA)scans of BubR1H/H ;INK-ATTAC mice confirmed that AP20187treatment prevented loss of adipose tissue

(Fig.2f).All major fat deposits were larger in AP20187-treated

BubR1H/H ;INK-ATTAC animals (Fig.2f)and individual adipocytes were markedly increased in size (Fig.2g).Consistent with this gen-erally increased adiposity,lateral skin contained significantly more

subdermal adipose tissue (Fig.2h).The above age-related phenotypes were not delayed upon AP20187treatment of BubR1H/H mice lacking INK-ATTAC (Fig.2b and Supplementary Fig.4).

Age-related phenotypes of BubR1H/H mice that arise in a p16Ink4a -independent fashion,such as cardiac arrhythmias and arterial wall stiffening 14,were not attenuated in AP20187-treated BubR1H/H ;INK-ATTAC mice (Supplementary Fig.5a,b).This correlated with lack of INK-ATTAC induction in heart and aorta (Fig.1c and Supplementary Fig.5c).Cardiac failure is presumably the main cause of death in BubR1H/H mice (data not shown),which could explain why the overall survival of AP20187-treated BubR1H/H ;INK-ATTAC mice was not substantially extended (Supplementary Fig.5d).To examine whether clearance of p16Ink4a -positive cells might have any overtly negative side effects,WT;INK-ATTAC mice were continuously treated with AP20187until 8months of age;however,no such effects were observed (data not shown).Taken together,these results indicate that continu-ous removal of p16Ink4a -expressing cells from BubR1H/H ;INK-ATTAC

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*Figure 2|BubR1H/H ;INK-ATTAC mice treated with AP20187from

weaning age on show delayed onset of p16Ink4a -mediated age-related

phenotypes.a ,Bone marrow cells cultured in rosiglitazone for 5days and then treated or not treated with AP20187(AP)for 2days before SA-b -Gal staining.Scale bar,50m m.b ,Incidence of lordokyphosis and cataracts.c ,Representative

images of 9-month-old mice.d ,Mean skeletal muscle fibre diameters of 10-month-old mice.ABD,abdominal muscle;Gastro,gastrocnemius muscle.e ,Exercise ability of 10-month-old AP20187-treated mice relative to age-matched untreated mice.Time is running time to exhaustion;distance is distance travelled at time of exhaustion;work is the energy expended to

exhaustion.f ,Body and fat depot weights of 10-month-old mice.Parentheses,s.d.Mes,mesenteric;Peri,perirenal;POV,paraovarian;SSAT,subscapular adipose tissue.g ,Average fat cell diameters in IAT of 10-month-old mice.h ,Dermis and subdermal adipose layer thickness of 10-month-old mice.Colour codes in e ,g and h are as indicated in d .Error bars,s.e.m.For all analysis n 56female mice per genotype (per treatment).*P ,0.05,**P ,0.01,***P ,0.001.

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Figure 3|AP20187-treated BubR1H/H ;INK-ATTAC mice have reduced numbers of p16Ink4a -positive senescent cells.a ,Images of SA-b -Gal stained IAT of 10-month-old mice.b –d ,Expression of senescence markers in IAT (b ),gastrocnemius (c )and eye (d )of 10-month-old AP20187-treated and untreated BubR1H/H ;INK-ATTAC-3mice relative to age-matched untreated WT;INK-ATTAC-3mice.Error bars indicate s.d.;n 53females per genotype per treatment.The expression of all genes is significantly decreased upon AP20187treatment (P ,0.05)with the exception of GFP in the eye.e ,BrdU incorporation rates in IAT and skeletal muscle.Error bars,s.e.m.;n 56females per genotype per treatment.*P ,0.05.

LETTER RESEARCH

00M O N T H 2011|V O L 000|N A T U R E |3

mice selectively delays age-related phenotypes that depend on p16Ink4a induction.

Next,we determined whether the delayed onset of age-related pathologies coincided with a reduction in the number of senescent cells in these tissues.The IAT of AP20187-treated BubR1H/H ;INK-ATTAC mice showed a marked decrease in SA-b -Gal staining com-pared with the IAT of untreated counterparts (Fig.3a).Corresponding decreases in other senescence-associated markers were also observed,as well as expected reductions in INK-ATTAC and GFP (Fig.3b and Supplementary Fig.6a).Skeletal muscle and eye had a similar reduc-tion in senescence indicators (Fig.3c,d and Supplementary Fig.6b,c).BrdU incorporation was lower in IAT and muscle tissue of untreated than treated animals (Fig.3e),supporting the contention that senescence-associated replicative arrest is decreased upon administra-tion of AP20187in BubR1H/H ;INK-ATTAC transgenic animals.Together,these data indicate that senescent cells were cleared from tissues and that this delays acquisition of age-related dysfunction in BubR1hypomorphic mice.

To investigate the effect of senescent cell clearance later in life when age-related phenotypes are apparent in BubR1H/H mice,we started AP20187treatment of BubR1H/H ;INK-ATTAC mice at 5months instead of weaning age and measured p16Ink4a -dependent age-related phenotypes at 10months.Cataracts had already fully maturated by the onset of AP20187treatment and remained unchanged (data not shown).Importantly,late-life treated animals had increased mean

muscle fibre diameters and showed improved performance in tread-mill exercise tests (Fig.4a,b).Furthermore,most fat depots of these animals were enlarged and adipocyte cell size and subdermal adipose layer thickness were significantly increased (Fig.4c–e).Senescence markers were substantially reduced in both fat and skeletal muscle

of AP20187-treated animals (Fig.4f,g and Supplementary Fig.7).

Analysis of 5-month-old untreated BubR1H/H ;INK-ATTAC-5mice revealed that the observed improvements in skeletal muscle and fat of late-life treated 10-month-old BubR1H/H ;INK-ATTAC-5mice reflect attenuated progression of age-related declines rather than a reversal of ageing (Fig.4a,c–e).Thus,late-life clearance of p16Ink4a -positive senescent cells attenuates progression of age-related decline in BubR1hypomorphic mice.Whether and how cellular senescence is related to age-related dis-eases,frailty and dysfunction has been one of the major open questions in the biology of ageing and clinical geriatrics 1.Here we present a novel transgenic mouse model that allows for the inducible removal of p16Ink4a -positive senescent cells.Remarkably,even though transcrip-tional regulation of endogenous p16Ink4a expression is highly complex,involving various transcriptional activators/repressors,epigenetic mechanisms and antisense non-coding RNA 19–22,we find that expres-sion of INK-ATTAC driven by a relatively small portion of the p16Ink4a promoter closely overlaps with that of endogenous p16Ink4a .By breed-ing INK-ATTAC mice into a progeroid mouse genetic background,

we show that both life-long and late-life clearance of the p16Ink4a -expressing senescent cells selectively delayed age-related pathologies

in tissues that accumulate these cells.Furthermore,our data indicate that acquisition of the senescence-associated secretory phenotype

(SASP),which enables cells to secrete a variety of growth factors,cytokines and proteases 4,contributes to age-related tissue dysfunction.

There were no overt side effects of senescent cell clearance in our model,even though it has been postulated that senescent cells enhance certain types of tissue repair 23,24.Our proof-of-principle experiments demonstrate that therapeutic interventions to clear senescent cells or block their effects may represent an avenue for treating or delaying

age-related diseases and improving healthy human lifespan.

METHODS SUMMARY

Mouse strains and drug treatments.The INK-ATTAC transgenic construct was made as follows.The FKBP–Casp8fragment was subcloned from the aP2-ATTAC transgenic construct 6(a gift from P.Scherer)and inserted into pBlueScriptII (Stratagene).A 2,617-bp segment of the murine p16Ink4a promoter was PCR amplified from BAC DNA to replace the aP2promoter.An IRES-EGFP fragment was inserted 39of the ATTAC.Transgenic founders were obtained by pronuclear injection of the INK-ATTAC construct into FVB oocytes.A PCR-based method was used for INK-ATTAC transgene identification (primer sequences are available upon request).BubR1H/H mice were generated as previously described 12.For AP20187(ARIAD Pharmaceuticals)treatments,animals were injected intraper-itoneally (i.p.)every 3days with 0.2m g g 21body weight of the dimer-inducing drug 6from weaning (‘life-long’)or 5-months on (‘late-life’).All mice were on a mixed 1293C57BL/63FVB genetic background.Animals were housed in a pathogen-free barrier environment throughout the study.The Institutional Animal Care and Use Committee approved experimental procedures on mice.

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F i b r e d i a m e t e r (μm )

Mouse (treatment) Weight (g) Fat (%) POV (g) Peri (g) IAT (g) Mes (g) SSAT (g) Brown (g)

10 mo H/H;ATTAC-3(–AP)17.3 (1.8) 14.8 (2.7) 0.044 (0.02) 0.007 (0.006) 0.058 (0.02) 0.048 (0.03) 0.040 (0.01) 0.066

(0.02) 10 mo H/H;ATTAC-3 (+AP after 5 mo)18.6 (2.6) 17.1 (3.3) 0.147* (0.13) 0.032* (0.026) 0.094*** (0.02) 0.091 (0.05) 0.095** (0.04) 0.080

(0.03)

10 mo H/H;ATTAC-5(–AP)17.6 (1.3) 14.3 (1.7) 0.080 (0.03) 0.017 (0.010) 0.070 (0.02) 0.081 (0.02) 0.061 (0.02) 0.071 (0.01)

10 mo H/H;ATTAC-5 (+AP after 5 mo)19.5 (2.1) 18.7* (3.8) 0.199** (0.08) 0.037* (0.016) 0.116* (0.05) 0.126 (0.07) 0.106* (.04) 0.100* (0.03) 5 mo H/H;ATTAC-5 (–AP)19.6 (3.0) 25.1 (3.5) 0.321 (0.21) 0.084 (0.05) 0.156 (0.08) 0.152 (0.08) 0.113 (0.06) 0.040

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NS NS NS 10 mo H/H;ATTAC-5

Figure 4|Treatment of older BubR1H/H

;INK-ATTAC mice with AP20187delays progression of p16Ink4a -mediated age-related phenotypes.a ,Mean skeletal muscle fibre diameters of the indicated mice.ABD,abdominal muscle;Gastro,gastrocnemius muscle.mo,months.b ,Improvement of exercise ability of the indicated mice relative to age-matched untreated mice.c ,Body and fat depots weights of the indicated mice.Parentheses,s.d.Mes,mesenteric;Peri,perirenal;POV,paraovarian;SSAT,subscapular adipose tissue.d ,Average size of fat cells in IAT of the indicated mice.e ,Subcutaneous adipose layer thickness of the indicated mice.f ,SA-b -Gal-stained IAT.g ,Expression of senescence markers in IAT and gastrocnemius of the indicated mice (n 53females per genotype per treatment).Expression of all genes,except those marked with NS,is significantly decreased (P ,0.05)upon late-life AP20187treatment.Colour codes in d and e are as indicated in a .Error bars indicate s.e.m.except in g where they indicate s.d.For analyses in a –f :n 555-month-old BubR1H/H ;INK-ATTAC-5–AP females;n 5910-month-old BubR1H/H ;INK-ATTAC-31AP and –AP females;n 5710-month-old BubR1H/H ;INK-ATTAC-51AP females;and n 5810-month-old BubR1H/H ;INK-ATTAC-5–AP females.*P ,0.05,**P ,0.01,***P ,0.001.NS,not significant.

RESEARCH LETTER

4|N A T U R E |V O L 000|00M O N T H 2011

Full Methods and any associated references are available in the online version of the paper at https://www.wendangku.net/doc/f615232985.html,/nature.

Received8May;accepted30September2011.

Published online2November2011.

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Supplementary Information is linked to the online version of the paper at

https://www.wendangku.net/doc/f615232985.html,/nature.

Acknowledgements We thank W.Zhou,D.Norris,T.Mann,U.Moedder,T.Pirtskhalava and S.Yamada for assistance;S.Khosla,T.von Zglinicki,L.Malureanu,R.Ricke and P.Galardy,and members of the https://www.wendangku.net/doc/f615232985.html,boratory for helpful discussions;and

P.Scherer for the gift of the aP2-ATTAC plasmid.This work was supported by the Ellison Medical Foundation(J.M.v.D.),the Noaber Foundation(J.M.v.D.and J.L.K.),the Robert and Arlene Kogod Center on Aging,and the National Institutes of Health(CA96985, J.M.v.D.and AG13925,J.L.K.).

Author Contributions D.J.B.,T.T.,J.L.K.,and J.M.v.D designed the INK-ATTAC strategy.

D.J.B.and T.W.performed most of the experiments,T.T.did the rosiglitazone experiments,N.K.L.and B.G.C.assisted with the analysis of muscle functionality and in vitro senescence,respectively,and B.v.d.S.helped supervise T.W.The manuscript was written by D.J.B.and J.M.v.D.All authors discussed results,made figures and edited the manuscript.J.M.v.D.directed and supervised all aspects of the study.

Author Information Reprints and permissions information is available at

https://www.wendangku.net/doc/f615232985.html,/reprints.The authors declare no competing financial interests. Readers are welcome to comment on the online version of this article at

https://www.wendangku.net/doc/f615232985.html,/nature.Correspondence and requests for materials should be addressed to J.M.v.D.(vandeursen.jan@https://www.wendangku.net/doc/f615232985.html,).

LETTER RESEARCH

00M O N T H2011|V O L000|N A T U R E|5

METHODS

Mouse strains and drug treatments.The INK-ATTAC transgenic construct was made as follows.The FKBP–Casp8fragment was subcloned from the aP2-ATTAC transgenic construct6(a gift from P.Scherer)and inserted into pBlueScriptII (Stratagene).A2,617-bp segment of the murine p16Ink4a promoter was PCR amp-lified from BAC DNA to replace the aP2promoter.An IRES-EGFP fragment was inserted39of the ATTAC.Transgenic founders were obtained by pronuclear injec-tion of the INK-ATTAC construct into FVB oocytes.A PCR-based method was used for INK-ATTAC transgene identification(primer sequences are available upon request).BubR1H/H mice were generated as previously described12.For AP20187 (ARIAD Pharmaceuticals)treatments,animals were injected intraperitoneally(i.p.) every3days with0.2m g g21body weight of the dimer-inducing drug6from weaning (‘life-long’)or5-months on(‘late-life’).All mice were on a mixed1293C57BL/63 FVB genetic background.Animals were housed in a pathogen-free barrier environ-ment throughout the study.The Institutional Animal Care and Use Committee approved experimental procedures on mice.

Statistical analysis.Prism software was used for the generation of all survival curves and statistical analyses.Two-tailed unpaired t tests were used for pairwise significance analysis in the following figures:Fig.1c,f and h;Fig.2d–h;Fig.3b–e; Fig.4a–e,g;Supplementary Fig.1b;Supplementary Fig.2;Supplementary Fig.3; Supplementary Fig.4;Supplementary Fig.5a–c;Supplementary Fig.6;and Supplementary Fig.7.Log-rank tests were used to determine overall and pairwise significance for incidence curves in Fig.2b and survival curves in Supplementary Fig.5d.For consistency in these comparisons,the following identifies the signifi-cance values:*P,0.05,**P,0.01,***P,0.001.

Cell culture.Culture of bone marrow cells was as previously described25.Briefly, tibia and femur bones of2-month-old WT;INK-ATTAC transgenic mouse lines were collected and flushed with DMEM containing15%FBS.After centrifugation at400g for10min and counting of viable cells with trypan blue,cells were resus-pended in DMEM containing15%FBS to a final concentration of53106viable cells per ml.Initially,cells were plated in6-well tissue culture dishes at3.5ml well21(1.93106cells cm22).Cultures were kept in a humidified5%CO2incubator at37u C for72h,when non-adherent cells were removed by changing the medium. Assays were performed on cells that had been trypsinized and seeded to confluency in24-well plates.To induce senescence and evaluate expression of the INK-ATTAC transgene,cells were treated with1m M rosiglitazone(Cayman Chemical Company) or with vehicle.The accumulation of GFP1cells was observed by fluorescence microscopy and transgene expression was verified by immunofluorescence staining for Flag(Origene)as described26.After5days of rosiglitazone treatment,cells were washed with PBS and treated with vehicle,1m M rosiglitazone,10nM AP20187,or both.After48h,cultures were fixed and stained for SA-b-Gal activity as described27. WT;INK-ATTAC MEFs were generated and cultured as previously described12.For induction of replicative senescence,WT;INK-ATTAC MEF cultures were main-tained in20%O2for12–15passages.For oncogene-induced senescence,early passage MEFs were infected with concentrated pBABE puro H-ras G12V retrovirus (Addgene plasmid9051)for48h.MEFs were then cultured in DMEM containing puromycin(2m g ml21)for5days.Cells from serial passage and H-ras induced senescence were sorted into GFP1and GFP–populations using a FACS Aria Cell Sorter(BD Biosciences)running FACSDiva software(serial passaging and H-ras expression yielded cultures with approximately90%and50%GFP1cells,respect-ively).Sorted cells were transferred to polyethylenimine-coated chambered slides and stained for SA-b-Gal according to manufacturer’s instructions(Cell Signaling).qRT–PCR and flow cytometry.RNA extraction,cDNA synthesis and qRT–PCR from whole-mouse tissue were performed as previously described15.To perform qRT–PCR on GFP1and GFP–cell populations of IAT,single-cell suspensions of stromal vascular fraction were prepared from,50mg IAT as described28.Cell sorting was performed as described above.RNA was extracted from the collected cells using an RNeasy Micro Kit(Qiagen)and cDNA synthesized using a WT-Ovation RNA Amplification kit(NuGEN Technologies),according to the manu-facturers’protocols.qRT–PCR primers were as follows:FKBP–Casp8forward, GAATCACAGACTTTGGACAAAGTT;FKBP–Casp8reverse,GGTCAAAGC CCCTGCATCCAAG;EGFP forward,CAAACTACAACAGCCACAA CG; EGFP reverse,GGTCACGAACTCCAGCAG.Sequences of other primers used were as previously described15.

Analysis of progeroid phenotypes.Bi-weekly checks for lordokyphosis and cataracts were performed as described15.Skeletal muscle fibre diameter measure-ments were performed on cross-sections of gastrocnemius and abdominal muscles of female mice as described15.Fifty total fibres per sample were measured using a calibrated computer program(Olympus MicroSuite Five).Fat cell diameter mea-surements were performed on IAT according to the same method.Dissection, histology and measurements of dermal and adipose layers of skin were performed as described previously12,although the lateral skin between the front and hind limb was used because this adipose layer is nearly three times thicker than dorsal skin. Measurements of body weight,length,gastrocnemius muscle and assorted adipose depots were performed on10-month-old females.Bone mineral content,bone mineral density and total body adipose tissue were analysed by DEXA scanning as previously described6.Exercise measurements were performed on10-month-old mice as previously described29.Animals were acclimated for3days for5min at a speed of5m min21before experimentation.For the experiment,the speed of the treadmill began at5m min21and was increased to8m min21after2min. Thereafter,the speed was increased at a rate of2m min21every2min and the time(in seconds)and distance(in metres)to exhaustion,as defined by an inability to move along the treadmill with stimulation,were determined.The formula to determine the amount of work(J)performed was:mass(kg)3g(9.8m s22)3 distance(m)3sin(h)(with an incline of h55u).Cardiac arrhythmia measure-ments were performed using a Vevo2100ultrasound system(Visualsonics)as previously described30.

In vivo BrdU incorporation and SA-b-Gal staining.Analyses for in vivo BrdU incorporation were performed in10-month-old female mice as described15. Adipose tissue depots were stained for SA-b-Gal activity as previously described12.

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cells from mouse bone marrow.Nature Protocols4,102–106(2009).

26.Malureanu,L.et al.Cdc20hypomorphic mice fail to counteract de novo synthesis

of cyclin B1in mitosis.J.Cell Biol.191,313–329(2010).

27.Dimri,G.P.et al.A biomarker that identifies senescent human cells in culture and

in aging skin in vivo.Proc.Natl https://www.wendangku.net/doc/f615232985.html,A92,9363–9367(1995).

28.Kirkland,J.L.,Hollenberg,C.H.&Gillon,W.S.Effects of fat depot site on

differentiation-dependent gene expression in rat preadipocytes.Int.J.Obes.Relat.

Metab.Disord.20(Suppl3),S102–S107(1996).

29.LeBrasseur,N.K.et al.Myostatin inhibition enhances the effects of exercise on

performance and metabolic outcomes in aged mice.J.Gerontol.A Biol.Sci.Med.

Sci.64A,940–948(2009).

30.Martinez-Fernandez,A.et al.iPS programmed without c-MYC yield proficient

cardiogenesis for functional heart chimerism.Circ.Res.105,648–656(2009).

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