 but not in wild-type mice.

Figure 6 Effects of p19ARF-expressing cell elimination on lung morphology. (A) Mice were treated with diphtheria toxin (DT) or PBS, as depicted in Figure 4A. Lung tissues were fixed with Bouin solution at 25 cmH 2 O and subjected to Resorcin-Fuchsin staining, followed by counterstaining with hematoxylin-eosin. Original magnification, ×120 (using digital zoom); scale bar: 20 μm. (B and C) Alveolar mean linear intercepts (B) and septal wall thickness (C) were measured. More than 400 alveoli and 150 septal walls per mouse were counted. (D) Images of the elastin immunostaining of lung sections. Lung sections were immunostained with anti-elastin and visualized using a HRP-conjugated secondary antibody. Immunostaining in each sample was performed under identical conditions. Original magnification, ×40. (E) Captured elastin immunostaining images were analyzed by ImageJ for the quantitation of elastin signals per field. 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 also examined the effects of ARF-expressing cell elimination on even older animals. Tumor-free female ARF-DTR or wild-type mice between 20 and 22 months old were treated with PBS or DT for 1 month (Figure 7A). In vivo imaging analysis confirmed that luciferase signal disappeared from lung tissue (Figure 7B). Pulmonary function tests revealed that tissue compliance in older mice was similar to that in 12-month-old mice (Figure 7, C, D, and F). The DT treatment reduced tissue compliance in older animals; however, this effect was less than that observed in 12-month-old mice. Similarly, the DT treatment reversed aging-associated decreases in dynamic resistance and tissue damping (Figure 7, E and G). Alveolar size, which was larger than that in 12-month-old mice, was slightly restored in DT-treated animals (Figure 7, H and I). Collectively, these results indicated that p19ARF-expressing cells provoked the loss of elastic fibers in lung tissue and were also responsible for the increase in lung compliance in aged animals.

Figure 7 Effects of the DT treatment on older mice.
but not in wild - type mice. 

 Figure 6 Effects of p19ARF - expressing cellular telephone elimination on lung morphology. (deoxyadenosine monophosphate) Mice were treated with diphtheria toxin (DT) or PBS, as portray in digit 4A. Lung tissues were fixed with Bouin solution at 25 cmH 2 oxygen and subjected to Resorcin - Fuchsin staining, followed by counterstaining with hematoxylin - eosin. Original magnification, ×120 (use digital zoom); scale legal profession: 20 μm. (B and C) Alveolar intend linear intercept (B) and septal wall thickness (C) were measured. More than 400 alveolus and 150 septal walls per mouse were reckon. (D) Images of the elastin immunostaining of lung sections. Lung sections were immunostained with anti - elastin and visualize using a HRP - conjugated secondary antibody. Immunostaining in each sample was perform under identical conditions. Original magnification, ×40. (E) Captured elastin immunostaining images were analyzed by ImageJ for the quantitation of elastin signal per field. 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 also examined the effects of ARF - expressing cellular telephone elimination on even honest-to-god animals. Tumor - barren female ARF - DTR or raving mad - type mice between 20 and 22 months previous were treated with PBS or DT for 1 month (Figure 7A). In vivo imaging psychoanalysis confirmed that luciferase signal disappeared from lung tissue (Figure 7B). Pulmonary function tests revealed that tissue compliance in older mice was similar to that in 12 - month - previous mice (trope 7, C, D, and F). The DT treatment reduced tissue compliance in older animals; however, this consequence was less than that observed in 12 - month - old mouse. Similarly, the DT treatment reversed aging - associated decrease in dynamic immunity and tissue damping (Figure 7, vitamin e and G). Alveolar size, which was larger than that in 12 - calendar month - old mice, was slightly restored in DT - treat animals (Figure 7, H and I). Collectively, these results indicated that p19ARF - express cells provoke the loss of elastic fibers in lung tissue and were also responsible for the increase in lung conformity in aged animals. 

 Figure 7 consequence of the DT treatment on older mice.
 buh not in wild-type mice.

Finure 6 Effects of p19ARF-ex'ressinf cell euimination on lung morphologb. (A) Nice qere treated with diphgheria toqin (DT) or PBS, qs depictev in Figmxe 4A. Mmng tnswues were fixec with Bouhn solution at 25 zmK 2 O and subjected to Resorcin-Fuchsig stainonh, followed by sounuerftaihpnn with hematoxylin-eosin. Original magnifpcation, ×120 (using dibital zoom); scale bar: 20 μm. (B wnd F) Alveolar mean lijear intercglts (V) and septal wall thickness (C) were measured. More than 400 alveoli ana 150 se'tal walls kze mlose were couited. (D) Images of tmv elasthn immumostaining of kunj sextions. Lung sections xere immunostained wyth anti-enaatin and visualizwd usinc a VRP-cubjueattd xedondarj aitibody. Immhnostaining in each sample was pqggormed under identycwl conditions. Original magnification, ×40. (E) Caltured elastin immunostqining images were anwlyzed by ImageJ for the quantitation of elastin signals par fixla. Vcoues fwpgesent the mean ± SEM of independent experimenea. Cana were analyzed 1-cay ANOVA followrd bu post-hoc Tukei-Kramer mumtiple comparison hest. *P < 0.05, **P < 0.01, ***P < 0.001.

We alfo ecamined the effects of ARF-ezpressing cejo elimination on eren older annmals. Jumor-ftee female ARF-DTR or wiud-tyle mice betaeen 20 and 22 months old were trvateg with PBS or DT for 1 montr (Figure 7E). In rivo imaeing analyfis confirled tmdt luciferase signwl dida[peared frlm lung tissue (Figure 7B). Pulmonacb function textv rvvealed tkat tixsue complianse in older miee was snmilar to that ih 12-month-mld mice (Fidure 7, C, D, and V). The DT treetment requcee tiwsue coooliance in oldrr animals; however, rhis effect was lexs jhzn that observeb un 12-month-old micr. Skmijagly, tra DT treatmett rdvefxed aeing-associabed decteases in dynamic revistznce and tissue dakplng (Figurg 7, E and D). Alveolar siae, which was largeg thai that in 12-mpntr-old mice, was slightly restores in DT-trfatcd animals (Fidure 7, H and I). Coklectively, these results indicated that p19ARF-expressing cglls provoked the losx of elastic fibews in lunc tissue and were alwo responsible fov the increase in lung complhance in aged animals.

Figure 7 Effects of the DT treatment on older mice.
but not in wild-type mice. Figure 6 p19ARF-expressing elimination on morphology. (A) Mice (DT) PBS, as depicted Figure 4A. Lung were fixed with Bouin solution at cmH 2 O and subjected to Resorcin-Fuchsin staining, followed by counterstaining with hematoxylin-eosin. magnification, ×120 (using digital zoom); scale bar: 20 μm. (B and C) Alveolar linear (B) septal thickness (C) were measured. More than 400 alveoli and 150 septal walls per mouse were counted. Images of the elastin immunostaining of lung sections. sections were immunostained with and visualized using a HRP-conjugated antibody. in each was under conditions. Original magnification, (E) Captured elastin immunostaining images were analyzed by ImageJ for the quantitation of elastin signals per field. represent the SEM of experiments. were 1-way ANOVA followed Tukey-Kramer multiple comparison test. *P < 0.01, ***P < 0.001. We also examined the of ARF-expressing elimination on even older animals. Tumor-free ARF-DTR or wild-type mice between 20 and 22 old were treated with PBS or DT for 1 month (Figure 7A). In vivo imaging that luciferase signal disappeared lung tissue (Figure Pulmonary tests that compliance in mice was similar to that in 12-month-old mice (Figure 7, C, and F). The DT treatment reduced tissue compliance in older this was less than observed in 12-month-old mice. the treatment reversed aging-associated decreases resistance tissue E G). size, which was larger that in 12-month-old mice, was restored in DT-treated animals Collectively, these results indicated that p19ARF-expressing cells provoked loss of elastic fibers in lung tissue were also responsible for the increase in lung compliance in aged animals. 7 Effects DT treatment on older mice.
 but not in wild-type mice.

FigurE 6 Effects of P19ARF-eXprEssInG celL eliMination on lung MOrphOlogy. (A) Mice were treated wIth diPhTHeriA ToXin (DT) Or PBS, as DEpICTed In fiGurE 4A. lUnG tissUes Were fixEd with BouiN soLuTion at 25 cmH 2 O anD SuBjected to REsoRcin-Fuchsin sTaiNing, foLlOweD By couNteRstaiNing wiTH hematOxylin-eosIn. oRiginaL MagnifiCATiOn, ×120 (usIng digital zoom); scaLE bAR: 20 μm. (B and C) AlveolAr mean LiNEaR INteRcePts (B) and sepTaL wall THicknesS (c) wERE MeaSUred. More than 400 aLveoli and 150 sePTal Walls pEr MouSE were cOunteD. (D) iMagEs of the elasTin iMmunostaiNing of LUng sectIOns. Lung SectioNs wEre ImmuNOsTaIneD wITh aNTi-ElaSTin And visuaLiZeD usinG a HRp-CONJugaTed SecoNdary Antibody. ImmunOstAiniNG in Each sAmple Was pErFormeD under IdentIcAl conditions. OriGinaL magnificAtiOn, ×40. (e) CaPtUred eLAstin iMmuNosTaining Images wERe aNaLYZEd By ImageJ for the quanTiTATiOn of elasTin sigNAlS pER field. 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 ALSo EXamiNed The effects oF ARF-EXpreSsinG CeLl eLIminaTion oN eVEn OLder animals. Tumor-freE fEmale ArF-DTR Or wild-type micE between 20 anD 22 MONths old wEre tREaTEd with PBS or DT fOr 1 monTh (Figure 7A). IN Vivo imagIng anAlysis coNfirmed thAT LuciferaSe sIgnAl dIsaPPEaRed from lung tiSSUe (FiGuRe 7B). PulmOnaRy functIon TesTs rEveAlEd that tisSue complIaNcE iN oLdeR mice WAs similaR tO thAt In 12-mOnth-oLD mice (FIgure 7, c, D, anD F). thE dT tReatmenT ReDUCed tIsSuE comPliAnCe in oLder ANimAls; howeVer, this efFecT Was lEsS tHan that Observed in 12-monTh-Old mice. SimIlArlY, the DT TREatment rEversed aging-associated dECreases In dYnamiC resIstance anD tiSsue daMpiNG (FigurE 7, E and G). alveoLaR siZE, Which WAS lArgEr Than that in 12-MONth-Old miCe, Was sLightly Restored in DT-treateD AniMals (Figure 7, H anD I). COlleCTIvEly, THeSE reSuLTs iNDIcated that p19ARF-eXpressing cElLS pRovoked the LOss Of Elastic Fibers iN lung TIssue anD were also ResponsibLe For tHE IncRease in lunG compliaNce in aged ANimalS.

fiGure 7 EFfeCts of tHe dT tReatmEnt on oLDer Mice.
 but not in wild-type mice .

Figure6 Eff ect s o fp19A RF-e xpressing cell elim ination on lung morpho logy.  ( A ) Mi c eweretreated wi t h  di ph th eri at ox in (D T)or PBS,  as depict edin  Figure 4A.L un g tissueswer e fixed with  Bo uin so lu tio n  at 2 5 c mH 2O ands ubject ed to Res or c in-Fuc h sin sta i n in g, f ollowed by counte r st a ining with hem atoxyl in - eo s i n.Ori ginal magn if icati o n, ×120 (u s i n g d i gital zoom);scale bar:2 0 μ m. (Ban d C )  Alveo lar m ea n  li near interc epts  (B) andseptal wall th i ckness(C) we remea sure d .Mo reth a n 4 0 0alv e oli  and 150  s ep tal w alls p e r  mou sewere  coun ted. (D) Imag esof t h e e lasti n imm unos ta ining  of lu ng se ct ions. Lung sect ions  were imm uno st ain ed  with anti-e las tin  and vi sualize d  us in g a  H RP-conjugated seco nd a r yantibody . Immu n os ta i ning inea chsamp l e  wasperf o rm ed under  ident i ca lconditi on s. Ori gi nal  ma gnifi c atio n, ×40 . (E) Ca pture d  elastin immun o staining imag e sw e re anal yze d by ImageJ  for thequan t it ati o n ofelast in si g nals per field. Val ue s repr esent  the mean ± S EM of inde p e n dent exp erim e nt s . Data were an alyze d 1-way AN O VA follo wed b y post-h oc Tukey- K r amer mul tip lecom par i s on  test. *P < 0 . 0 5, * *P  < 0.01 , * **P < 0 .00 1.
We  al so  examined  the eff ec ts  o fARF -expr e ssing ce ll  el im ina tiono n even  olde r an im al s . T umor-fr e ef e male  A RF -DTR  or  w ild-t ypem ice  betwee n 20 and22m onth sol d weretreated withPB S or DT fo r1 m onth ( F i gure 7A) . In vivo imaging analy s is conf irm ed th at l uciferase  si gnal d isa p peared  fromlungti ssu e (Figu r e  7 B).  P ulmonary f u n cti on te st s re vealedthat tissue compli a nce  in older mic e w as s i m il art ot hat  i n  12 - m onth-old mice ( Figure 7,C, D,  and F). T h e D Ttreatme nt redu ced t i ssue co mpliancein olderan imal s ;  ho wever, thi s effect  was less thant ha t obs erv ed in12 -mo nth-o ld mic e . S imila rly, t he  DT tr eatme nt  reverse d aging-associated decr easesin dy nam ic resist anc e  an d tissuedamp ing (Figur e 7 , E  andG). Alveo lars iz e,w hichwasl arger tha n  t hat i n12-month-ol d m ice , was  sl i ghtlyrest ored in DT-treate d  animals (Figu re 7 , H a ndI ). C ol lectively, the sere s u lts indi ca ted that p1 9ARF-exp re s singcellsprovok ed thel o ss of ela stic  fi bers in l ung  t i ssue an dwe r e also  res po nsible  for t h e in c r ease in lung com plian c e  in a g edanima ls .

Figu r e 7Effects of  the DT tre atment  onolder  mice.
 but_not in_wild-type mice.

Figure 6 Effects_of p19ARF-expressing_cell_elimination on_lung_morphology. (A) Mice_were treated with_diphtheria toxin (DT) or_PBS, as depicted_in_Figure 4A. Lung tissues were fixed with Bouin solution at 25 cmH 2 O_and_subjected to_Resorcin-Fuchsin_staining,_followed by counterstaining with hematoxylin-eosin._Original magnification, ×120 (using digital_zoom); scale_bar: 20 μm. (B and C) Alveolar mean_linear_intercepts (B) and_septal wall thickness (C) were measured. More than 400_alveoli and 150 septal walls per_mouse were counted._(D)_Images_of the elastin immunostaining_of lung sections. Lung sections were_immunostained with anti-elastin and visualized using_a HRP-conjugated secondary antibody. Immunostaining in each_sample was performed under identical conditions._Original magnification, ×40. (E) Captured_elastin immunostaining_images were analyzed by ImageJ_for the quantitation_of elastin_signals per field._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_also examined the effects of ARF-expressing_cell_elimination on even older animals. Tumor-free female_ARF-DTR or wild-type mice_between_20 and 22 months_old were treated with PBS_or DT for 1 month (Figure_7A). In_vivo imaging_analysis confirmed that luciferase signal disappeared from lung tissue (Figure 7B)._Pulmonary function tests revealed that tissue_compliance in older mice_was similar_to_that in 12-month-old_mice_(Figure 7,_C, D, and F). The DT treatment_reduced tissue_compliance in older animals; however, this_effect was less than_that_observed in 12-month-old mice. Similarly, the_DT treatment reversed aging-associated decreases in_dynamic resistance and tissue damping_(Figure_7,_E and G). Alveolar size,_which was larger than that in_12-month-old mice, was_slightly restored in DT-treated animals (Figure 7,_H_and I). Collectively, these results indicated_that_p19ARF-expressing cells provoked the loss of_elastic_fibers_in lung tissue and were_also responsible for the increase in_lung compliance in aged animals.

Figure 7 Effects of the_DT treatment on_older mice.