 successfully removed from lung tissue of ARF-DTR mice (Figure 4, C and D).

Figure 4 Downregulation of senescence genes by eliminating p19ARF-expressing cells. (A) Experimental design. Twelve-month-old female wild-type or ARF–diphtheria toxin receptor (ARF-DTR) mice were intraperitoneally injected with diphtheria toxin (DT) or PBS twice with a 2-week interval. Two weeks after the second DT injection, mice were sacrificed and their lungs were analyzed. (B) Luciferase signals in the lung area were measured in ARF-DTR mice using Living Image in vivo imaging software. Values represent the mean ± SEM of independent experiments. (C and D) Total RNA was extracted from the lungs of 2-month-old and 12-month-old female ARF-DTR mice treated with DT or not treated for 4 weeks (C) or 12-month-old wild-type mice treated or not treated with DT for 4 weeks (D). The expression of ARF, INK4a, and p21 was analyzed by real-time PCR. mRNA levels were normalized to GAPDH in each mouse. Values represent the mean ± SEM of independent experiments. Data were analyzed 1-way ANOVA followed by post-hoc Tukey-Kramer multiple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

We then performed pulmonary function tests on these mice. As previously reported (20, 21), static lung compliance (Cst) was significantly higher in older animals than in young animals (Figure 5, A and B). The DT treatment resulted in the marked recovery of lung elasticity (decrease in Cst) in ARF-DTR mice but not in wild-type mice. Similarly, the DT treatment reversed aging-associated changes in dynamic compliance, dynamic resistance, tissue elastance, and tissue damping in ARF-DTR mice but not in wild-type mice (Figure 5, C–F). These results clearly indicated that the p19ARF-expressing cells that accumulated in 12-month-old lung tissue had deleterious effects on pulmonary function and that aging-associated declines in pulmonary function were ameliorated by the elimination of these p19ARF-expressing cells.

Figure 5 Restoration of lung function by eliminating p19ARF-expressing cells. (A) Pressure-volume curves of ARF–diphtheria toxin
successfully removed from lung tissue of ARF - DTR mice (Figure 4, C and D). 

 name 4 Downregulation of aging genes by eliminating p19ARF - expressing cell. (A) Experimental design. Twelve - calendar month - old female wild - type or ARF – diphtheria toxin sense organ (ARF - DTR) mouse were intraperitoneally injected with diphtheria toxin (DT) or PBS doubly with a 2 - week interval. Two weeks after the second DT injection, mice were sacrifice and their lungs were analyzed. (B) Luciferase signals in the lung area were measure in ARF - DTR mice using Living Image in vivo imagination software. Values represent the mean ± SEM of autonomous experiments. (degree centigrade and D) Total RNA was extracted from the lungs of 2 - month - old and 12 - calendar month - old female ARF - DTR mice treated with DT or not treated for 4 weeks (C) or 12 - calendar month - erstwhile wild - type mice treated or not treated with DT for 4 weeks (D). The expression of ARF, INK4a, and p21 was analyzed by actual - time PCR. mRNA levels were anneal to GAPDH in each mouse. Values represent the mean ± SEM of independent experiment. Data were analyzed 1 - way ANOVA followed by post - hoc Tukey - Kramer multiple comparison trial. * P < 0.05, * * P < 0.01, * * * P < 0.001. 

 We then performed pulmonary function test on these mice. As previously reported (20, 21), static lung compliance (Cst) was importantly higher in old animal than in young animals (Figure 5, A and B). The DT treatment resulted in the marked recovery of lung elasticity (decrease in Cst) in ARF - DTR mouse but not in wild - type mice. Similarly, the DT treatment revoke aging - consort changes in dynamic conformity, dynamic electric resistance, tissue elastance, and tissue muffle in ARF - DTR mice but not in barbarian - type mice (Figure 5, C – F). These results clearly bespeak that the p19ARF - expressing cell that accumulated in 12 - month - old lung tissue had deleterious effects on pneumonic function and that aging - associate declines in pulmonary function were ameliorated by the elimination of these p19ARF - expressing cells. 

 Figure 5 Restoration of lung routine by eliminating p19ARF - expressing cells. (A) Pressure - volume curve of ARF – diphtheria toxin
 sufcessfully removed from uung tissue of CEF-DTR kice (Fjgure 4, C and D).

Figure 4 Downregulation od sentfcence genes by elkminating p19ARF-expeessmng cells. (A) Expecjmental desigh. Twente-month-old femake wild-typa or ARF–diphtharka toxin receptor (ARF-DTR) mice were ineraperiyojeally injecteq wiuh qiphfheria toxin (DT) or PBS twice with z 2-week pnterval. Two weekx after the second DT injeftioj, mice were sacriflced and thgjr jyngs were anxlyzed. (B) Lleiferase sifnals in the lung area were mearured in ARF-DTR muce osing Living Imagv in vivo imanpng sofdware. Vslues represenb the meqn ± SEM of independenv experiments. (C and Q) Total RTA was extracted frim the nungv of 2-nongh-omd ahd 12-monhh-omd female ZRF-DTR mice treated with DT or njn treated for 4 weekf (S) or 12-month-old wild-type mice treated or tot treated with DT for 4 wweks (D). The expression of ARF, IGK4a, and p21 was analyzed by real-time PCR. mRNA levelv werx vornajkzef to GAPDH in each mouse. Values represent the jesn ± SEM of indepcndent experiments. Dwts were analyzea 1-way CHOBA followed by posh-hoc Tuhey-Krqmer multyple comparison test. *P < 0.05, **P < 0.01, ***P < 0.001.

We then peryorned pulmonary funccion tests ou thesg mice. As previously reported (20, 21), sfatic lung fompliancs (Cst) was signifizannly vigher in older animals thwn in youig annmals (Fieure 5, A anq B). The DT treabkent resulted in tje matked racovery of lung elasticity (decrease in Csv) in ARF-DTR moca blt not in wild-bype mice. Similwrly, the DT trgatment rzversea aging-asskciated changes in dynamic compnlance, dynamir resistagce, rissye elasgxnce, and tissur damping in ARF-DTR mice but not in wlld-tyke mice (Figure 5, C–Y). Uhwse results clraruy ynfirated dhat the p19ARX-expfesrong cdlls that agcuoulayed in 12-month-old lung tisaue had deleterioux cffects ob pulmonwry function snd that aging-assofiatev declmnes im polmonary function were ameliorafed by thf ejimination os thcse k19ARF-expressnng cells.

Figure 5 Restoration of lung funrtion by eliminating p19AEF-expressing cells. (C) Kressure-volumx curvqs of ARF–giphtheria toxin
successfully removed from lung tissue of ARF-DTR 4, and D). 4 Downregulation of cells. Experimental design. Twelve-month-old wild-type or ARF–diphtheria receptor (ARF-DTR) mice were intraperitoneally injected diphtheria toxin (DT) or PBS twice with a 2-week interval. Two weeks after second DT injection, mice were sacrificed and their lungs were analyzed. (B) Luciferase in lung were in ARF-DTR mice using Living Image in vivo imaging software. Values represent the mean ± SEM independent experiments. (C and D) Total RNA was from the lungs of and 12-month-old female ARF-DTR mice with or not for weeks or 12-month-old wild-type treated or not treated with DT for 4 weeks (D). The expression of ARF, INK4a, and p21 analyzed by mRNA levels normalized GAPDH each mouse. Values mean ± SEM of independent experiments. 1-way ANOVA followed by post-hoc Tukey-Kramer multiple comparison *P < **P < 0.01, ***P < 0.001. then performed pulmonary function tests on these mice. previously reported (20, 21), static lung compliance (Cst) was significantly higher in older animals than animals (Figure 5, A B). The DT resulted the recovery lung elasticity in Cst) in ARF-DTR mice but not in wild-type mice. Similarly, DT treatment reversed aging-associated changes in dynamic compliance, dynamic resistance, and damping in ARF-DTR but not in wild-type (Figure C–F). These results clearly the cells 12-month-old tissue deleterious effects on pulmonary and that aging-associated declines in function were ameliorated by cells. Figure 5 Restoration of lung function by p19ARF-expressing cells. (A) Pressure-volume curves of ARF–diphtheria
 successfully removed from luNg tissue of aRF-DTr miCe (FIgUre 4, C And D).

figure 4 DownreguLAtioN of senescence genes by elIminaTiNG p19ARf-ExPressIng cellS. (a) EXPEriMeNtAl dEsIGn. twelvE-moNth-old fEmale wild-tYpe Or aRF–diphtheriA ToXin receptoR (ARf-DTR) mice were IntRaperiToNeaLLy injEctEd witH diphtHEria toXin (DT) or PBs tWIce witH A 2-week inTERvAl. TwO weeks after the secONd dt injection, mice Were saCrIFiCED anD thEir lungs weRe AnalyZEd. (B) LuciFErASE SigNAls in the lung aRea were measURed In ARF-DtR MicE Using LIving imAGe iN vivo imaginG sofTware. ValuEs reprESent the MEan ± SEM oF indepEndEnt ExpeRImEnTs. (C AnD d) ToTAl rNA WAs eXtracted FrOm The luNgs oF 2-MONTh-olD anD 12-monTh-old Female ARF-DTR mIce TreaTEd wIth DT Or not TreaTeD for 4 wEeks (C) oR 12-montH-oLd wild-type mice tReatEd or not trEatEd WitH Dt for 4 wEEks (D). ThE exPreSsion of aRF, INK4a, ANd p21 WaS ANAlYzed by real-time PCR. mrNa LEvEls were nOrmaliZEd To gaPDH in eaCh MouSe. VaLUEs repReseNT tHe mean ± SEm of indEPeNdEnt expeRiMents. DAtA weRe aNalyzED 1-way aNOVA fOllowed bY post-HOc Tukey-Kramer mULtiple compariSOn TESt. *p < 0.05, **p < 0.01, ***P < 0.001.

We TheN performed pUlmoNAry fUnctIOn TesTS on thEse miCe. aS pREviously reported (20, 21), staTiC lung cOmpliAnce (Cst) was sigNificantly HIGHer in oldEr anIMaLS than in young anImals (figure 5, A and b). the DT treAtmenT resulteD in the marKED recoverY of LunG elAstICItY (decrease in CsT) IN ARF-dTr mice buT noT in wild-TypE miCe. SImiLaRly, the DT tReatment ReVeRsEd AgiNg-assOCiated chAnGes In DynAmic cOMplianCe, dynAmic ReSiSTanCe, tissuE ElASTancE, aNd TissUe dAmPing iN ARF-dtR mIce but nOt in wild-tYpe MIce (FIgUrE 5, C–F). ThesE results clearLy Indicated tHaT thE p19ARF-eXPRessing cElls that accumulated in 12-moNTh-old luNg tIssue Had dEleteriouS efFects oN puLMonary FunctiOn and ThAt aGINg-assOCIaTed DeClines in puLMOnaRy funCtIon wEre amelIorated by the eliminATioN of these p19ARF-eXprEssiNG CeLls.

fIgURe 5 REsTOraTIOn of lung functioN by eliminaTiNG p19aRF-expressINg cElLs. (A) PresSure-volUme cuRVes of ARf–diphtherIa toxin
 successfully removed from  lung tiss ue of  AR F-D TR  mic e (F igure 4, C and D).
Figure 4 Downregulati on of  s e nesc e nc e gen es by e l im i n ati ng  p 19A RF - ex press ing  cells.  (A) Exper ime nt al design. T w el ve-month-o ldfemale wild- typ e or A RF –di p hther iatoxin  recep t or (AR F-DTR) mi ce were i n traperi t o ne ally  injected with di p ht h eria toxin (DT ) or P BS tw i c e w ith  a 2-weekin terva l . Two w e ek s a fte r  the second D T injection ,  mi ce wer esac r ificed  andth e irlungs wereanal yzed. (B)  Lucif e rase si g nals in  the l ung  ar ea w e re  m eas ur e d i n  A RF- D TRmice usi ng  L iving  Ima g e i n vi voimag ing s oftware. Valu esrepr e sen t the  mean  ± S EM  of i ndepen dentex periments. (C a nd D ) Total R NAwa s e xt racte d  fromthe  lu ngs of2-month - old  a n d 12 -month-old femaleAR F - DT R mice t reated wi th DT or no ttre ated f or 4week s  ( C) or 12 -month - ol dwild-ty pe  micetr eat edor no t  tre ated w ith DT f or 4w eeks (D). Thee xpression ofA RF , IN K 4a,and  p21 was an alyz e d by  rea l -t ime PCR.mRNAle v el s  were normalized to  G APDH i n eac h mouse. Valu es represe n t the mean  ± S E Mo f independentexper iments. Da t a were a nalyz ed 1-way  ANOVA fo l l owed bypos t-h ocTuk e y -K ramer multipl e comp ar ison te st.  *P < 0 .05 , * *P< 0 .0 1, ***P <  0.001.
W eth en  pe rform e d pulmon ar y f un cti on te s ts onthese  mic e.  A s  pr eviousl y  r e p orte d(2 0, 2 1),  s tatic  lun g  co mplianc e (Cst) w ass igni fi ca ntly hi gher in older  a nimals tha ninyounga n imals (F igure 5, A and B). TheD T treat men t res ulte d in themar ked re cov e ry oflung e lasti ci ty( d ecrea s e  i n C st ) in ARF-D T R  mi ce bu tnotin wild -type mice. Simila r ly,  the DT treat men t re v e rs eda gi n g-a ss o cia t e d changes in dy namic comp li a nc e, dynamic res is tance,tissueelast a nce, an d tissuedamping i nARF- D T R m ice but no t in wil d-type mi c e (Fi g ur e 5,C–F ). The se  re sults  clear l y i ndica ted th at  the p 19ARF -e xpressin g cells that accumulate d in 1 2-mon th- old lungtis s uehad delet erio us effects  on  pu lmona ryf uncti on a n dtha t  agin g-as s ociated d e cl ine s in  pulmonaryf u n cti on we rea melior ated  by the eliminati o n of these p19 ARF- e x pre ssi n g ce ll s.

Figure 5 R est or a t ion of l un g functionby elimi na t ing p 19ARF- expres sing ce l l s. (A) Pr essu re- volume cu rve so f ARF–d ip ht h eria t oxin 
 successfully_removed from_lung tissue of ARF-DTR_mice (Figure_4,_C and_D).

Figure_4 Downregulation of_senescence genes by_eliminating p19ARF-expressing cells. (A)_Experimental design. Twelve-month-old_female_wild-type or ARF–diphtheria toxin receptor (ARF-DTR) mice were intraperitoneally injected with diphtheria toxin (DT)_or_PBS twice_with_a_2-week interval. Two weeks after_the second DT injection, mice_were sacrificed_and their lungs were analyzed. (B) Luciferase signals_in_the lung area_were measured in ARF-DTR mice using Living Image in_vivo imaging software. Values represent the_mean ± SEM_of_independent_experiments. (C and D)_Total RNA was extracted from the_lungs of 2-month-old and 12-month-old female_ARF-DTR mice treated with DT or not_treated for 4 weeks (C) or_12-month-old wild-type mice treated or_not treated_with DT for 4 weeks_(D). The expression_of ARF,_INK4a, and p21_was analyzed by real-time PCR. mRNA_levels were normalized_to GAPDH in each mouse. Values_represent_the mean ±_SEM_of_independent experiments._Data were analyzed_1-way_ANOVA followed_by_post-hoc Tukey-Kramer multiple comparison test. *P_<_0.05, **P < 0.01, ***P < 0.001.

We_then performed pulmonary function_tests_on these mice. As_previously reported (20, 21), static_lung compliance (Cst) was significantly higher_in older_animals than_in young animals (Figure 5, A and B). The DT treatment_resulted in the marked recovery of_lung elasticity (decrease in_Cst) in_ARF-DTR_mice but not_in_wild-type mice._Similarly, the DT treatment reversed aging-associated changes_in dynamic_compliance, dynamic resistance, tissue elastance, and_tissue damping in ARF-DTR_mice_but not in wild-type mice (Figure_5, C–F). These results clearly indicated_that the p19ARF-expressing cells that_accumulated_in_12-month-old lung tissue had deleterious_effects on pulmonary function and that_aging-associated declines in_pulmonary function were ameliorated by the elimination_of_these p19ARF-expressing cells.

Figure 5 Restoration of_lung_function by eliminating p19ARF-expressing cells. (A)_Pressure-volume_curves_of ARF–diphtheria toxin