1
Au ophagy main ains s emness by p e en ing senescence
Lau a Ga cía-P a 1, Ma a Ma ínez-Vicen e2^, Eusebio Pe digue o1, Lau a O e 1, Ja ie
Rod íguez-Ub e a3, Elena Rebollo4, Vanessa Ruiz-Bonilla1, Susana Gu a a1, Es eban
Balles a 3, An onio L. Se ano1, Ma co Sand i5^ and Pu a Muñoz-Cáno es1,6*
1Cell Biology G oup, Depa men o Expe imen al and Heal h Sciences, Pompeu Fab a
Uni e si y (UPF), CIBER on Neu odegene a i e diseases (CIBERNED), E-08003
Ba celona, Spain; 2Neu odegene a i e Diseases Resea ch G oup, Vall d'Heb on
Resea ch Ins i u e-CIBERNED, E-08035 Ba celona, Spain; 3Ch oma in and Disease
G oup, Cance Epigene ics and Biology P og amme (PEBC), Bell i ge Biomedical
Resea ch Ins i u e (IDIBELL), E-08907 L'Hospi ale de Llob ega , Ba celona, Spain;
4Molecula Biology Ins i u e o Ba celona (IBMB-CSIC), 08028 Ba celona, Spain;
5Depa men o Biomedical Science, Uni e si y o Pado a, 35100 Pado a, I aly. Tele hon
Ins i u e o Gene ics and Medicine (TIGEM), 80131 Napoli, I aly; 6ICREA, Ba celona,
Spain.
^Equal con ibu ion
*Co esponding au ho :
Pu a Muñoz-Cáno es (pu a.munoz@up .edu )
2
SUMMARY
Du ing aging, muscle s em cell egene a i e unc ion declines. A ad anced ge ia ic age,
his decline is maximal due o ansi ion om a no mal quiescence in o an i e e sible
senescence s a e. How sa elli e cells main ain quiescence and a oid senescence du ing
hei long li e emains la gely unknown. He e we epo ha basal au ophagy is
indispensable o main ain he s em-cell quiescen s a e in mice. Failu e o au ophagy in
physiologically aged sa elli e cells o i s gene ic impai men in young cells causes
senescence en y by loss o p o eos asis, inc eased mi ochond ial dys unc ion and
oxida i e s ess, esul ing in nume ical and unc ional sa elli e cell decline. Au ophagy
ees ablishmen e e ses senescence and es o es egene a i e unc ions in ge ia ic
sa elli e cells. Since au ophagy also declines in human ge ia ic sa elli e cells, hese
indings unco e au ophagy as a decisi e s em cell- a e egula o and ha e implica ions
o os e ing muscle egene a ion in sa copenia.
3
The egene a i e capaci y o skele al muscle elies on long-li ed Pax7-exp essing
muscle s em cells (called sa elli e cells), which a e no mally in quiescence (a G0
e e sible a es s a e). In esponse o issue damage, hese cells ac i a e, en e he cell
cycle and ei he expand and o m new myo ibe s o sel - enew o es o e he quiescen
sa elli e cell pool1-4. Quiescence he e o e appea s as a simple way o unc ionally
main aining he s em cell popula ion h oughou li e in he absence o egene a i e
demand, pa icula ly in issues wi h li le u no e , such as skele al muscle.
Sa copenia, he age- ela ed loss o skele al muscle mass and unc ion, is maximal
a ge ia ic age. A his las s age o li e, skele al muscle shows a p o ound egene a i e
impai men ha con ibu es o he indi idual’s physical incapaci a ion. Recen s udies ha e
demons a ed ha aged skele al muscles ail o e ain s em cell quiescence5-7. Bo h he
numbe and he unc ionali y o muscle s em cells decline wi h aging5-10, wi h sa elli e cells
swi ching om a quiescence o a p e-senescence s a e in sa copenic muscle a ge ia ic
age6. How sa elli e cells main ain quiescence du ing hei long li e and a oid acquisi ion o
he senescence p og am un il ad anced age is la gely unknown.
Using physiologically aged mice, we show ha quiescen muscle s em cells
p ese e hei in eg i y o e ime h ough ac i e main enance o o ganelle and p o ein
homeos asis (p o eos asis) as cellula quali y con ol mechanism. In pa icula , we
demons a e ha hese do man s em cells display con inuous basal mac oau ophagy
( om now on ‘au ophagy’; i.e. he p ocess o deg ada ion o long-li ed p o eins and
damaged o ganelles in lysosomes11,12). Wi h aging, his ac i i y declines. Physiological
decline o au ophagy in old sa elli e cells, o i s gene ic impai men in young cells, esul s
in oxic cellula was e accumula ion, which p o okes senescence en y.
Ou esul s indica e ha muscle s em cells p ese e hei G0- e e sible quiescence
s a e om en e ing a G0-i e e sible senescence s a e h ough au ophagy. Impo an ly,
gene ic and pha macological egimes ha eins all basal au ophagy in ge ia ic mice
e e sed s em cell senescence and es o ed egene a ion. These indings ha e
implica ions o egene a i e medicine in sa copenia.
4
RESULTS
Quiescen muscle s em cells basal au ophagy is impai ed in aging
We in e oga ed he quiescen sa elli e cell ansc ip ome o changes in
p o eos asis genes (as compa ed o ac i a ed cells)13-15 and unco e ed au ophagy as he
mos p e alen pa hway in he quiescen s a e (Ex ended Da a Fig. 1a; Supplemen a y
Table 1). K-means clus e ing analysis e ealed an age-associa ed down egula ion o
au ophagic genes in quiescence (Ex ended Da a Fig. 1b; Supplemen a y Table 1).
Au ophagy is an e olu iona y conse ed p ocess o sel -deg ada ion o cellula
componen s (o ganelles, cy osol po ions and mis olded p o eins) by au ophagosomes
which a e deli e ed o he lysosomal machine y, hus p e en ing was e accumula ion11,12,
and which has been implica ed in aging o di e en model o ganisms11,12,16,17. To
in es iga e he occu ence o au ophagy in quiescen muscle s em cells we used GFP-
LC3 (a well-known ma ke o au ophagosomes) ansgenic mice18,19. Quiescen sa elli e
cells we e luo escence-ac i a ed cell so ing (FACS)-isola ed (Ex ended Da a Fig. 1c)
om es ing muscle o young (3 mon hs) and old GFP-LC3 mice (20-24 mon hs). Punc a e
GFP-LC3-s aining was ound in young cells, being inc eased in old cells (Fig. 1a;
Ex ended Da a Fig. 1d; Supplemen a y Video 1-2). We nex used he au ophagy- lux
inhibi o Ba ilomycin (Ba ), ha p e en s lysosome deg ada ion, hus inc easing punc a e
GFP-LC3 exclusi ely when au ophagy is ac i e20, as a p oxy o au ophagic ac i i y. A
a iance wi h young cells, Ba ea men demons a ed he old sa elli e cells’ incapaci y o
u he au ophagosome o ma ion, as shown by GFP-LC3 luo escence le els (Fig. 1b).
These esul s indica e, i s , cons i u i e au ophagic ac i i y in young quiescen sa elli e
cells; and, second, impai ed ac i i y du ing aging. Fluo escence, ansmission-elec on
mic oscopy and Wes e n-blo ing analyses con i med in old sa elli e cells he accumula ion
o au ophagic esicles (Ex ended Da a Fig. 1e, 1 ), p62 (a p o ein egula ing au ophagic
clea ance o dys unc ional o ganelles/agg ega es)-con aining agg ega es, ubiqui in (Ub)-
posi i e inclusions (Ex ended Da a Fig. 1g), inc eased p62 le els and educed LC3II-
accumula ion a e Ba ea men (Ex ended Da a Fig. 1h, 1i) -common ai s o de icien
au ophagy. O no e, 2-week- ea men wi h Rapamycin (o Spe midine), well-known
au ophagy-inducing egimes21,22, in old mice es o ed s em-cell basal au ophagy (Fig. 1c;
Ex ended Da a Fig. 1j; Supplemen a y Video 3 and 4), educing p o ein and o ganelle
agg ega es (Fig. 1d; Ex ended Da a Fig. 1k).
Au ophagy ees ablishmen a oids senescence and es o es egene a ion in
ge ia ic s em cells
Sa elli e cells a ge ia ic age (o e 28 mon hs in mice) en e a senescen s a e6,23.
We in es iga ed whe he dys egula ed basal au ophagy may unde lie loss o bona- ide
quiescence. Using a mRFP–GFP–LC3 cons uc 24 ( andem- luo escen - agged LC3
epo e ), ans ec ed in young, old and ge ia ic sa elli e cells, in combina ion wi h Ba , we
ound a highe blockade o au ophagic lux in ge ia ic han old cells, wi h espec o young
cells. In he absence o Ba , ed-colo -labeled LC3 punc a (ma u e au olysosomes) we e
only abundan in young cells. Ba ea men induced yellow-colo -labeled LC3 punc a
(non- used au ophagosomes) accumula ion in young cells, which was lowe o blun ed in
old and ge ia ic cells (Fig. 2a). Ge ia ic sa elli e cells also showed inc eased
colocaliza ion o p62-ubiqui in agg ega es in non-deg aded au ophagosomes (Fig. 2b). As
p62 ma ks damaged o ganelles o deg ada ion by selec i e au ophagy, while Ub ma ks
subs a es o hei deg ada ion by bo h he ubiqui in-p o easome sys em (UPS) and
5
selec i e au ophagy, he inc eased signal o bo h p o eins and hei colocaliza ion
demons a es ha he de ec on au ophagy in hese cells is due, a leas in pa , o a block
in au ophagosomes/lysosomes clea ance.
To in es iga e i es o ing au ophagy could escue he cell-in insic i e e sible cell-
cycle and egene a i e block o ge ia ic cells, we eng a ed eshly-isola ed GFP-labeled
young and ge ia ic sa elli e cells (p e- ea ed wi h Rapamycin o con ol- ehicle) in o p e-
inju ed muscles o young- ecipien mice. Au ophagy eac i a ion signi ican ly es o ed
expansion o ge ia ic cells (exp essing Pax7, Ki67, MyoD o Mgn) a e 4-day eng a men
(Fig. 2c; Ex ended Da a Fig. 2a-c) and p e en ed senescence (ge ocon e sion), as shown
by p16INK4a and H2AX educ ion (Ex ended Da a Fig. 2d). Rapamycin (o Spe midine)
ea men also dec eased ge ia ic senescen cells (β-galac osidase-posi i e, SA-β-gal+)
(Ex ended Da a Fig. 2e, 2 ) and e-es ablished p oli e a ion (Ex ended Da a Fig. 2e). Mo e
impo an ly, gene ic-au ophagy e ueling, by o e exp essing A g7 (c ucial o
au ophagosome o ma ion) (Fig. 2c; Ex ended Da a Fig. 2g, 2h) escued he p oli e a i e
de ec , while educing senescence (Ex ended Da a Fig. 2e). Fu he mo e, sa elli e cell
ansplan a ion and whole-muscle g a expe imen s demons a ed ha he sole A g7
in oduc ion in ge ia ic sa elli e cells escued hei in insic egene a i e capaci y, allowing
new muscle- ibe o ma ion (Fig. 2c; Ex ended Da a Fig. 2i, 2j).
Gene ic au ophagy inhibi ion in young quiescen sa elli e cells causes senescence
en y
To in es iga e i basal au ophagy dys up ion causally b eaks quiescence, we
in e c ossed A g7- loxed mice wi h Pax7-C e and Pax7-C eER mice, o impai au ophagy
cons i u i ely (A g7ΔPax7) o inducibly (A g7ΔPax7ER) a e amoxi en adminis a ion).
In e c ossing A g7ΔPax7 wi h GFP-LC3 mice (A g7ΔPax7:GFP-LC3) con i med loss o
au ophagosomes in quiescen A g7-null sa elli e cells (Ex ended Da a Fig. 3a, 3b).
Impo an ly, he sa elli e cell pool was se e ely educed in A g7ΔPax7 mice (Fig. 3a;
Ex ended Da a Fig. 3c). Tamoxi en adminis a ion o 3-mon h-old A g7ΔPax7ER mice led o
sa elli e cell loss a e 30 days (Fig. 3b), indica ing ha basal au ophagy is equi ed o
bo h es ablishmen and main enance o he adul quiescen s em cell popula ion.
Remaining A g7ΔPax7ER sa elli e cells showed unexpec ed induc ion o p16INK4a, p21CIP1 and
p15INK4b, and DNA damage (H2AX+ cells) –signs o p ema u e aging (Fig. 3c, 3d;
Ex ended Da a Fig. 3d). O no e, A g7ΔPax7 sa elli e cells did no unde ake mi o ic o
myogenic di e en ia ion pa hways (Ex ended Da a Fig. 3e). Thus, loss o au ophagy wi h
aging may causally unde lie he age-associa ed muscle s em cell nume ical decline5,6,8-
10,25.
In esponse o muscle inju y, Pax7+ cells om young A g7ΔPax7 mice showed
educed ac i a ion and expansion capaci y (Fig. 3e; Ex ended Da a Fig. 3 ), and
accele a ed en y in o deep senescence23,26 (ge ocon e sion27) in i o and in i o, as
demons a ed by: SA-β-gal+-s aining (Fig. 3 ), H2AX+, p16INK4a+ and phospho yla ed-S6+
cells, and egene a i e ailu e, shown by educed cell p oli e a ion and size o
egene a ing ibe s (Fig. 3g; Ex ended Da a Fig. 3g-l). Con i ming he cell-in insic
egene a i e ailu e, ewe GFP+ ibe s de i ed om A g7-null sa elli e cells we e ound in
ansplan a ion expe imen s (Ex ended Da a Fig. 3m, 3n), and his ailu e could no be
escued by Rapamycin (no Spe midine) (Ex ended Da a Fig. 3m-o).
6
Mi ophagy ailu e and ROS-induc ion d i e senescence in au ophagy-de icien
sa elli e cells
How could loss o au ophagy in young quiescen sa elli e cells induce p ema u e
aging? Gene ic au ophagy impai men in sa elli e cells caused apid accumula ion o
p62/Ub-posi i e agg ega es, and mi ochond ia and lysosomes (mi o acke , and
lyso acke and Lamp1), as in aged cells (Fig. 4a, 4b; Ex ended Da a Fig. 4a). The e was
also a lowe p opo ion o heal hy mi ochond ia in old (and A g7 ΔPax7ER
) sa elli e cells, as
e ealed by educed memb ane po en ial (lowe a io TMRM/Mi oT acke G een MFI) (Fig.
4a, 4b). Fu he mo e, mi ophagy (cellula capaci y o clea by au ophagy damaged
mi ochond ia) was de ec i e in ge ia ic sa elli e cells, as indica ed by mi ochond ia
accumula ion inside au ophagosomes/lysosomes ( h ough mi ochond ia/lysosomes
(TOM20/Lamp1) colocaliza ion) (Ex ended Da a Fig. 4b). In i o Rapamycin (o
Spe midine) ea men o ge ia ic mice es o ed mi ophagy in sa elli e cells (Ex ended
Da a Fig. 4b- ). Consis en wi h age-impai ed mi ophagy, young, bu no ge ia ic, cells,
we e capable o elimina ing CCCP-damaged mi ochond ia (Ex ended Da a Fig. 4d, 4e).
How does al e ed mi ophagy lead o sa elli e cell senescence wi h aging? We
de ec ed highe le els o eac i e-oxygen species (ROS), Pa kin (ma king damaged
mi ochond ia o deg ada ion by mi ophagy), and DNA-damage ma ke s in A g7-de icien
sa elli e cells (Ex ended Da a Fig. 5a, 5b; Fig. 3d, 3g; Ex ended Da a Fig. 3h), associa ed
o p16INK4a and pS6 induc ion (Fig. 3g; Ex ended Da a Fig. 3g; Ex ended Da a Fig. 4h).
Highe ROS and ROS-mi ochond ia colocaliza ion we e also obse ed in ge ia ic sa elli e
cells, co ela ing wi h impai ed mi ophagic lux (Fig. 5a; Ex ended Da a Fig. 4g). O no e,
Ba -induced au ophagy block caused g ea e mi ochond ia accumula ion in young cells,
compa ed o ge ia ic and A g7ΔPax7ER cells, pa alleling ROS inc ease (Fig. 5b). To add ess
hei ole, ROS we e inhibi ed wi h T olox ( i amin E analog) (Ex ended Da a Fig. 5c).
T olox ea men o old GFP-LC3 mice inc eased GFP-LC3 punc a (a e Ba - ea men )
and educed p62/Ub agg ega es and mi ochond ia-ROS colocaliza ion in GFP-LC3
sa elli e cells (Ex ended Da a Fig. 4g; Ex ended Da a Fig. 5d). A enua ion o au ophagic
block by ROS inhibi ion was u he con i med in Ba - ea ed aged cells h ough LC3-II
accumula ion (Ex ended Da a Fig. 5e, 5 ) and mRFP-GFP-LC3 andem- epo e , which
de ec ed educed au ophagosomes (RFP+/GFP+ punc a) and escued au ophagic lux
(Ex ended Da a Fig. 5g). Impo an ly, T olox ea men p e en ed appea ance o
senescence ma ke s (Fig. 5d, 5e), es o ed expansion (Fig. 5d), and escued he cell-
in insic p oli e a i e/ egene a i e de ec o ge ia ic sa elli e cells a e ansplan a ion (Fig.
5 ; Ex ended Da a Fig. 5h). Thus, inc eased ROS, esul ing om impai ed au ophagy,
d i e sa elli e cell senescence in aged cells.
Loss o he polycomb ep essi e complex-1 (PRC1)-media ed H2A
monoubiqui ina ion o lysine119 (H2Aub) a INK4a locus d i es p16INK4a induc ion in
ge ia ic sa elli e cells6 (Ex ended Da a Fig. 5i). We ound ha T olox ea men es o ed
INK4a locus H2Aub modi ica ion in ge ia ic and A g7-de icien sa elli e cells (Fig. 6a, 6b),
esul ing in p16INK4a ep ession, and his educed senescence while p omo ing p oli e a ion
(Fig. 5c-e; Ex ended Da a Fig. 5j; Fig. 6c). No ably, p16INK4a gene ic-silencing wi h sho -
hai pin-RNA es o ed p oli e a ion in A g7ΔPax7 sa elli e cells while educing senescence-
associa ed genes and SA-β-gal+ cells, and augmen ing hei egene a i e capaci y
(Ex ended Da a Fig 5k; Ex ended Da a Fig. 6a, 6b). Thus, he ROS-induced p16INK4a axis
links impai ed au ophagy and senescence in aging sa elli e cells.
Human ge ia ic sa elli e cells exhibi au ophagy de ec s ha p omo e senescence
7
Skele al muscles om ge ia ic indi iduals show sa copenia and p esence o
senescen sa elli e cells (Ex ended Da a Fig. 6c, 6d)6. As in mice, human sa elli e cells
om ge ia ic indi iduals showed de ec i e p o ein- and o ganelle-clea ance, as e idenced
by p62 and mi ochond ial accumula ion (Ex ended Da a Fig. 6e, 6 ) compa ed o young
cells, which was igh ly-associa ed wi h inc eased ROS le els (Ex ended Da a Fig. 6 , 6g)
and SA-β-gal+ cells (Ex ended Da a Fig. 6h), consis en wi h educed p oli e a i e po en ial
(Ex ended Da a Fig. 6i). The causal ole o impai ed au ophagy on he ge ocon e sion o
aging human sa elli e cells unde p oli e a i e p essu e was sus ained by he capaci y o
Rapamycin o e e he abno mal mi ochond ial con en , p o ein agg ega es and ROS
(Ex ended Da a Fig. 6e, 6 ), and senescence pheno ype (Ex ended Da a Fig. 6h-k). Thus,
es o a ion o au ophagy and o ganelle homeos asis in aged human sa elli e cells su ices
o escue senescence, as in mu ine sa elli e cells.
8
DISCUSSION
In issues wi h li le u no e , e e sible quiescence is he no mal s em-cell s a e
h oughou li e. Howe e , quiescence is known o be p og essi ely los wi h aging due o
sys emic/niche- and in insic- ac o al e a ions2,5. Recen s udies showed ha a ge ia ic
age, he no mal s em-cell quiescen s a e is subs i u ed by an i e e sible senescence
s a e, which esul s in nume ical and unc ional s em cell decline6. The mechanisms
accoun ing o main enance o quiescence, p ese a ion o he s em cell pool and
p e en ion o senescence du ing an indi idual’s li e emain la gely unknown. Ou esul s
demons a e ha quiescen sa elli e cells a e equipped wi h cy op o ec i e and cellula
quali y con ol mechanisms ha ac i ely ep ess he senescence p og am, he eby
p ese ing cells’ in eg i y and i ness. We p o ide e idence o loss o au ophagy in sa elli e
cells wi h aging, esul ing in accumula ion o damaged p o eins and o ganelles, leading o
senescence and s em cell exhaus ion. Consis en wi h his, gene ic inhibi ion o au ophagy
speci ically in sa elli e cells o young mice caused apid senescence en y, esul ing in
s em-cell nume ical and unc ional exhaus ion, and de ec i e muscle egene a ion. These
indings came as a double-su p ise conside ing ha basal-au ophagy decline in quiescen
s em cells o physiologically aged mammalian o ganisms was no desc ibed be o e, and
ha au ophagy was no mally conside ed an e ec o pa hway, a he han a cause, o
senescence, pa icula ly in oncogene-induced senescence28-31.
How can au ophagy balance quiescence and senescence in muscle s em cells?
He e we show ha in adul es ing muscle, quiescen s em cells a enua e p o eo oxici y
by main aining high basal au ophagy lux, cons i u ing a homeos a ic “clean up” p ocess.
This unc ion is pa icula ly c i ical in a non-di iding s em cell, whe e mi o ic dilu ion o
in acellula oxic deb is does no ake place17,32. Au ophagy ailu e in aged es ing s em
cells leads o accumula ion o damaged p o eins and dys unc ional o ganelles, specially
mi ochond ia, which gene a es enhanced ROS le els ha cause DNA damage and
senescence en y, consis en wi h p e ious s udies33-39. Indeed, we unco e ROS as a key
epigene ic egula o o he senescence-p omo ing gene p16INK4a in aging s em cells, by
impeding PRC1-media ed lysine119 H2A-ubiqui ina ion, he equi ed epigene ic ma k o
INK4a locus silencing. Consis en wi h his, ea men o ge ia ic mice (and mice wi h
sa elli e cell-speci ic A g7 de iciency) wi h an ioxidan s no only es o ed PRC1-media ed
INK4a locus ep ession and p e en ed sa elli e cell senescence, bu also es o ed
egene a i e capaci y. Impo an ly, signs o impai ed au ophagy and loss o p o eos asis,
co ela ing wi h senescence and de ec i e myogenic unc ions, we e also obse ed in
human sa elli e cells om ge ia ic indi iduals.
A a iance wi h ou indings, a ecen elegan s udy demons a ed ha , upon in
i o s ess, au ophagy does no decline, bu is e en induced in hema opoie ic s em cells
(HSCs) wi h aging, consis en wi h main enance o HSC numbe 40. Thus, we p opose ha
long-li ed quiescen s em cells wi hin low u n-o e issues p ima ily ely on au ophagy o
p ese e i ness and a oid senescence, and ha s em cells o skele al muscle pa icula ly
lose his p o ec ion du ing aging (Ex ended Da a Fig. 6 ). O in e es , a ecen s udy also
epo ed he need o au ophagy o ac i a ion o young sa elli e cells41.
Ou s udies hus demons a e ha au ophagy is a decisi e ac o be ween he
quiescence and senescence a e o muscle s em cells (Ex ended Da a Fig. 6l). Al hough
aging-induced senescence is o en iewed as an inescapable and i emediable p ocess,
we p o ide e idence ha in i o es o a ion o cons i u i e au ophagy (o neu aliza ion o
excessi e ROS) a e s in acellula damage accumula ion, and p e en s sa elli e cell
9
senescence and unc ional decline in old mice, as well as in aged human s em cells,
ein o cing he no ion ha he in insic-aging clock in s em cells can be pha macologically
manipula ed.
16
in ensi y o each e en (in a e age) o he selec ed cell popula ion, in he chosen
luo escence channel.
Whole ansc ip ome analysis o FACS so ed sa elli e cells
FACS so ed sa elli e cells we e collec ed in lysis bu e and RNA ex ac ion was
pe o med using RNeasy Mic o ki (Qiagen). cDNA was used on a ansc ip ome analysis
by Agilen Su eP in G3 Mouse GE 8x60K high densi y mic oa ay slides, pe o med a he
mic oa ay Uni o CRG (Ba celona, Spain). Mic oa ay analysis was pe o med wi h 3
animals each. Da a was no malized using cyclic loess, and di e en ially exp essed genes
we e iden i ied using AFM 4.044 o all pai wise compa isons. Raw da a was aken om he
Fea u e Ex ac ion ou pu iles and was co ec ed o backg ound noise using he no mexp
me hod. To assu e compa abili y ac oss samples quan ile no maliza ion was used.
Di e en ial exp ession analysis was ca ied ou on non con ol p obes wi h an empi ical
Bayes app oach on linea models (limma). Resul s we e co ec ed o mul iple es ing
acco ding o he False Disco e y Ra e (FDR) me hod. S a is ical analysis was pe o med
wi h he Bioconduc o p ojec (h p://www.bioconduc o .o g/) in he R s a is ical
en i onmen . Venn diag ams we e gene a ed using BioVenn45.
In i o ea men s
Au ophagy o aged C57BL/6 and GFP-LC3 mice was induced as ollows: one g oup o
mice was i.p. injec ed wi h 4mg/kg BW Rapamycin (LC Labo a o ies) o ehicle (DMSO)
e e y o he day o 2 weeks; a second g oup was i.p. injec ed wi h 30mg/kg BW T olox (6-
hyd oxy-2,5,7,8- e ame hylch oman-2-ca boxylic acid, Sigma) o ehicle (DMSO) daily o
2 weeks; and he hi d g oup o mice was ea ed wi h 3mM Spe midine (S2626 Sigma) in
d inking wa e o 2 weeks.
Sa elli e cell eng a men
Sa elli e cell ansplan s we e pe o med as in Sousa-Vic o e al., 20146, ollowing an
adap ed p o ocol46. Basically, quiescen FACS-isola ed sa elli e cells we e collec ed, e-
suspended in 20% FBS HAM’S F10 medium and injec ed in o muscles o ecipien mice
p e iously inju ed wi h CTX he day be o e. Recipien mice we e SCID mice. Fo each
mouse, 10,000 cells we e injec ed. A 4 days ( o p oli e a ion, senescence analyses) o 1
mon h (muscle egene a ion) a e cell injec ions, eng a ed muscles we e collec ed and
p ocessed o muscle his ology. Resul s a e exp essed as % o GFP+ cells coun ed pe
muscle sec ion (being 100% he young cells con ol da a).
In i o ea men s
Expe imen s o in i o escue o de ec i e au ophagy in sa elli e cells we e pe o med in
20% FBS-con aining HAM’S F10 medium (G ow h medium), and adding ei he Rapamycin
(100ng/ml, LC Labo a o ies), T olox (25μl/ml, Sigma), Spe midine (5μM, Sigma) o ehicle
(DMSO) o 48 h. Mi ochond ial, lysosomal, and ROS analyses o ChIP expe imen we e
pe o med igh a e ea men s, while p oli e a ion assay (B dU s aining) and senescence
analysis (SA-β-gal assay and de e mina ion o RNA, p o ein exp ession o senescence
ma ke s), we e pe o med 96 h a e ea men s.
Sa elli e cell ea men s o in i o eng a men in inju ed muscles: F eshly FACS isola ed
sa elli e cells om es ing muscle o young and ge ia ic mice we e ea ed o 48 h wi h
Rapamycin (100ng/ml, LC Labo a o ies), T olox (25μl/ml, Sigma) o ehicle (DMSO) p io
o eng a men in o p e-inju ed muscles o ecipien mice. Fo each mouse, 10,000 cells
17
we e injec ed. A 4 days a e cell injec ions, eng a ed muscles we e collec ed and
p ocessed o muscle his ology.
Ba ilomycin (10nM Sigma B1793) was used o block au ophagy o 4 h a 37ºC and o
analyze au ophagosome accumula ion by FACS, immunos aining and Wes e n blo ing.
CCCP (Ca bonyl cyanide 3-chlo ophenylhyd azone) (10μM Sigma C2759), which
abolishes he link be ween he espi a o y chain and he phospho yla ion sys em in in ac
mi ochond ia, causes mi ochond ia uncoupling and was used o ea sa elli e cells in i o
o 1 h o induce he selec i e au ophagy o CCCP-damaged mi ochond ia (mi ophagy).
Plasmid ans ec ion
F eshly isola ed cells we e ans ec ed wi h mRFP-GFP-LC320 plasmid using
Lipo ec amine 3000 (In i ogene), and u he ea ed o 48h wi h T olox (25μl/ml, Sigma)
o ehicle (DMSO) o analysis on glass slides (The mo Scien i ic 177402). Cells we e
ixed wi h 4% pa a o maldehyde (PFA) in PBS o 10 minu es and nuclei we e s ained wi h
DAPI (In i ogen). A e washing, glass slides we e moun ed wi h Mowiol. Measu ing
au ophagy lux h ough his me hod is based on he concep o lysosomal quenching o
GFP. GFP is a s ably olded p o ein and ela i ely esis an o lysosomal p o eases.
Howe e , he low pH inside he lysosome quenches he luo escen signal o GFP, which
makes i di icul o ace he deli e y o GFP-LC3 o lysosomes. In con as , RFP exhibi s
mo e s able luo escence in acidic compa men s, and mRFP-LC3 can eadily be de ec ed
in au olysosomes. By exploi ing he di e ence in he na u e o hese wo luo escen
p o eins (i.e., lysosomal quenching o GFP luo escence e sus lysosomal s abili y o RFP
luo escence), au ophagic lux can be mo phologically aced wi h an mRFP-GFP-LC3
andem cons uc 20. Wi h his andem cons uc , au ophagosomes and au olysosomes a e
labeled wi h yellow (i.e., mRFP and GFP) and ed (i.e., mRFP only) signals, espec i ely.
P oli e a ion assay
Sa elli e cells we e labeled wi h B dU (1.5 μg ml−1; Sigma) o 1 h. B dU-labeled cells we e
de ec ed by immunos aining using a an i-B dU an ibody (Ox o d Bio echnology; 1:500)
and a speci ic seconda y bio inyla ed goa an i- a an ibody (Jackson Inmuno esea ch;
1:250). An ibody binding was isualized using Vec as ain Eli e ABC eagen (Vec o
Labo a o ies) and DAB. B dU-posi i e cells we e quan i ied as pe cen age o he o al
numbe o cells analyzed.
Senescence-associa ed β-galac osidase ac i i y
Senescence-associa ed β-galac osidase (SA-β-gal) ac i i y was de ec ed in sa elli e cells
using he Senescence β-Galac osidase S aining ki (Cell signaling), acco ding o he
manu ac u e ’s ins uc ions. SA-β-gal-posi i e cells we e quan i ied as pe cen age o he
o al numbe o cells analyzed.
Len i i us in ec ion
F eshly isola ed sa elli e cells we e ex i o in ec ed wi h dis inc len i i us o 12 h.
Medium was eplaced and cells we e ansplan ed in o inju ed muscle o ecipien mice o
in i o analysis, o subjec ed o in i o assays. LV-A g7, used o A g7 o e exp ession in
sa elli e cells, was kindly p o ided by Elieze Masliah’s labo a o y47. LV-sh p16INK4a, used o
silence p16INK4a, and LV-sh Sc amble (used as con ol), we e p e iously desc ibed in
Sousa-Vic o e al, 20145.
18
He e og a ing expe imen s
Ex enso digi o um longus (EDL) muscles om ge ia ic WT mice we e in ec ed wi h
Len i i us (LV-A g7 o LV-GFP, as well as LV-shp16INK4a o LV-sh Sc amble) and g a ed
immedia ely on o he ibialis an e io (TA) muscle o young WT ecipien mice, and
egene a ion ( o ma ion o new myo ibe s de i ed om EDL-associa ed sa elli e cells) in
he ansplan ed EDL muscles was analyzed a e 6 o 8 days. Fibe size o eMHC+
myo ibe was analyzed using Fiji p og am.
RT–qPCR: RNA ex ac ion, cDNA syn hesis and PCR
To al RNA was isola ed om ei he FACS-isola ed sa elli e cells o mouse muscle issue
o human myoblas s ob ained om human muscle biopsies, using T ipu e eagen (Roche
Diagnos ic Co po a ion) o RNeasy Mic o ki (Qiagen), and analysed by RT–qPCR. Fo
qPCR expe imen s, DNase diges ion o 10 mg o RNA was pe o med using 2 U DNase
(Tu bo DNA- ee, Ambion). Complemen a y DNA (cDNA) was syn hesized om o al RNA
using he Fi s -S and cDNA Syn hesis ki (Ame sham Biosciences). Real- ime PCR
eac ions we e pe o med on a Ligh Cycle 480 Sys em using Ligh Cycle 480 SYBR
G een I Mas e eac ion mix (Roche Diagnos ic Co po a ion) and speci ic p ime s.
The mocycling condi ions we e as ollows: ini ial s ep o 10 min a 95 °C, hen 50 cycles o
15 s dena u a ion a 94 °C, 10 s annealing a 60 °C and 15 s ex ension a 72 °C. Reac ions
we e un in iplica e, and au oma ically de ec ed h eshold cycle (C ) alues we e
compa ed be ween samples. T ansc ip o he ibosomal p o ein L7 housekeeping gene
was used as endogenous con ol, wi h each unknown sample no malized o L7 con en .
The ollowing p ime s we e used:
Gene Fo wa d p ime Re e se p ime
p16INK4a CATCTGGAGCAGCATGGAGTC GGGTACGACCGAAAGAGTTCG
p21CIP1 CCAGGCCAAGATGGTGTCTT TGAGAAAGGATCAGCCATTGC
MyoD GCCGCCTGAGCAAAGTGAATG CAGCGGTCCAGGTGCGTAGAAG
Mgn GGTGTGTAAGAGGAAGTCTGTG TAGGCGCTCAATGTACTGGAT
Ki67 ACCGTGGAGTAGTTTATCTGGG TGTTTCCAGTCCGCTTACTTCT
p15INK4b TCTTGCATCTCCACCAGCTG CTCCAGGTTTCCCATTTAGC
A g7 TCTGGGAAGCCATAAAGTCAGG GCGAAGGTCAGGAGCAGAA
Elec on mic oscopy images
Fo elec on mic oscopy images, ibialis an e io (TA) muscles om 3 and 24 mon h-old
WT mice we e ixed wi h 2% pa a o maldehyde/2,5% glu a aldehyde in phospha e bu e
(0.1M-pH7.4). Samples we e p ocessed by he CCi Mic oscopy Facili y a he Uni e si y
o Ba celona. Images we e acqui ed using a Jeol 1010 mic oscope, wo king a 80 k and
equipped wi h a CCD Mega iew III came a. Iden i ica ion o sa elli e cells in skele al
muscle by elec on mic oscopy was based on cell size, con en o he e och oma in and
19
posi ion wi h espec o basal lamina.
Wes e n blo ing
P epa a ion o mouse and human sa elli e cell lysa es and Wes e n blo ing was
pe o med as desc ibed p e iously by Pe digue o e al. 200748. An ibodies used we e:
an i-p62/SQSTM1 an ibody p oduced in abbi (Sigma P0067), abbi an i-LC3 (No us
Biologicals NB100-2331), phospho-S6 ibosomal p o ein (Se 240/244) XPTM Rabbi mAb
(Cell Signaling 5364), abbi an i-p16 (San a C uz Bio echnology sc-1207), abbi an i-
Pa kin (Abcam ab15954), S6 ibosomal p o ein (54D2) mouse (Cell Signaling 2317),
γH2AX Se 139 (Cell Signaling 2577S), abbi an i-53BP1 (Abcam ab21083) and Tubulin
(Sigma T-6199).
Ch oma in Immunop ecipi a ion (ChIP)
B ie ly, eshly isola ed sa elli e cells we e cul u ed wi h T olox o ehicle (DMSO) o 48 h
and c oss-linked wi h 1% o maldehyde o 15 min a oom empe a u e. Fo each ChIP,
300,000 cells we e lysed in 130 L o Lysis Bu e B (Low Cell ChIP Ki , Diagenode) and
ch oma in was sonica ed o 10 min in a M220 Focused-ul asonica o ™, Co a is (Du y
cycle 5%, Peak inciden powe 75 Wa s and 200 cycles pe bu s ). Sonica ed ch oma in
was hen dilu ed and subjec ed o immunop ecipi a ion wi h 3 L o an ibody agains
Ubiqui yl-His one H2A (Lys119) (D27C4) (Cell Signaling, 8240) o 3 L o IgG. Bound
ac ion and inpu we e analyzed by qPCR using speci ic p ime se s o he INK4a locus.
Gene
INK4a_RD
INK4a_exon1
INK4a_exon2
Fo wa d p ime
GGTCTCCCCTAGCAGGA
TTC
CCGGAGCCACCCATTAA
ACTA
CCCAACACCCACTTGAG
GAA
Re e se p ime
GCCTGTCATTAAACAGGGTGA
CAAGACTTCTCAAAAATAAGACACT
GAAA
CAGAGGTCACAGGCATCGAA
His ology and immunohis ochemis y in muscle c yosec ions
Tibialis an e io (TA) and ex enso digi o um longus (EDL) muscles we e ozen in
isopen ane cooled wi h liquid ni ogen, and s o ed a −80 °C un il analysis. 10 μm sec ions
we e collec ed om muscles and we e ei he s ained wi h hema oxylin/eosin (HE) o
immunos ained. Labelling o c yosec ions wi h mouse monoclonal p ima y an ibodies was
pe o med using he pe oxidase o luo escein M.O.M ki s aining (Vec o Labo a o ies)
acco ding o he manu ac u e ’s ins uc ions. Double immunos aining was pe o med by
sequen ial addi ion o each p ima y and seconda y an ibody using app op ia e posi i e
and nega i e con ols. Sec ions we e ai d ied, ixed on PFA 2–4%, washed on PBS and
incuba ed wi h p ima y an ibodies acco ding o manu ac u e ’s ins uc ions a e blocking
o 1 h a oom empe a u e wi h a high p o ein-con aining solu ion on PBS (Vec o
Labo a o ies). Subsequen ly, he slides we e washed on PBS and incuba ed wi h
app op ia e seconda y an ibodies and labelling dyes. Fo immuno luo escence, seconda y
an ibodies we e coupled o Alexa-488, Alexa-568 o Alexa-647 luo och omes, and nuclei
we e s ained wi h DAPI (In i ogen). A e washing, issue sec ions we e moun ed wi h
20
Mowiol.
An ibodies used o immunohis ochemis y
Immunohis ochemis y on muscle c yosec ions o isola ed sa elli e cells was pe o med
wi h he ollowing an ibodies: GFP (In i ogen A6455 and A es labs GFP-1020), an i-
eMHC (F1.652), an i-Pax7 (DSHB), p16 (San a C uz sc-1207), γH2AX Se 139 (2577S),
abbi polyclonal an i-MyoD (San a C uz Bio echnology sc-760), an i-myogenin (DSHB
F5D), poly ubiqui inyla ed p o eins, mul i ubiqui in chains, mouse mAb (Enzo li e sciences
PW8805), an i-p62/SQSTM1 an ibody p oduced in abbi (Sigma P0067), mouse
monoclonal an ibody o LC3 (NanoTools 5F10), LAMP-1 (San a c uz Bio echnology
sc-19992), phospho-S6 ibosomal p o ein (Se 240/244) XPTM Rabbi mAb (Cell Signaling
5364), an i-CD56 (BD Pha mingen 556325), an i-TOM20 (ab56783).
Human muscle samples
Muscle biopsies om eigh adul s and en ge ia ic (28 ± 7 and 83 ± 7 yea s old,
espec i ely) human subjec s we e ob ained ia he Tissue Banks o Resea ch om Vall
d’Heb on and San Joan de Deu Hospi als and especially ia he EU/FP7 Myoage
Conso ium. Muscle biopsies we e aken om he as us la e alis muscle unde local
anaes hesia (2% lidocaine). A po ion o he muscle issue was di ec ly ozen in mel ing
isopen ane and s o ed a −80 °C un il analysis. Human p ima y myoblas s om 5
young/adul (25 ± 4 yea s old) and 5 ge ia ic (75 ± 4 yea s old) subjec s we e ob ained
om he EU/FP7 Myoage Conso ium o pu chased om Cook Myosi e and cul u ed
ollowing he p o ided ins uc ions.
Digi al image acquisi ion and p ocessing
Digi al images we e acqui ed using: (1) an up igh mic oscope DMR6000B (Leica)
equipped wi h a DFC300FX came a o immunohis ochemical colo pic u es and a
Hamama su ORCA-ER came a o immuno luo escence pic u es; (2) con ocal images o
muscle sec ions o isola ed sa elli e cells we e aken using ei he a Zeiss LSM-780
con ocal sys em wi h a Plan-Apoch oma 63x/1.4 oil objec i e o a Leica SPE con ocal
lase scanning mic oscope sys em wi h HCX PL Fluo a 10×/0.30, 20×/0.50 and 40×/0.75
objec i es. The di e en luo opho es (3 o 4) we e exci ed by using he 405, 488, 568 and
633 exci a ion lines. Acquisi ion was pe o med using Zeiss LSM so wa e Zen Black o
Leica Applica ion o LAS AF so wa e (Leica). Images we e composed and edi ed in
Pho oshop CS5 (Adobe), whe e backg ound was educed using b igh ness and con as
adjus men s applied o he whole image. To assess myo ibe size, indi idual ibe s we e
manually ou lined and hei c oss-sec ional a ea (CSA) was de e mined wi h he public
domain image analysis so wa e Fiji. Fluo escence in ensi y o selec ed p o eins o each
cell was quan i ied using Fiji so wa e and he a e age o ela i e luo escence was
exp essed as MFI.
The numbe and pe cen age o cellula a ea occupied by GFP-LC3 punc a we e
de e mined on digi al images wi h Fiji and he cell image analysis so wa e CellP o ile 49.
Colocaliza ion o RFP-LC3 and GFP-LC3 punc a was de e mined on he maximum
p ojec ion o h ee Z-sec ions using a Fiji au oma ed mac o pipeline calcula ing single and
double-posi i e au ophagosomes. Colocaliza ion o p62/Ub was de e mined on digi al
images Fiji, acco ding o50, wi h espec o he o al cellula a ea. The Pea son’s coe icien
( ) was used o analyze he co ela ion o he in ensi y alues o g een and ed pixels in
21
dual-channel images. This coe icien measu es he s eng h o he linea ela ionship
be ween he in ensi ies in wo images calcula ed by linea eg ession and anges om 1 o
−1, wi h 1 s anding o comple e posi i e co ela ion and −1 o a nega i e co ela ion, wi h
ze o s anding o no co ela ion49. Video econs uc ions o au ophagosomes we e
gene a ed in Ima is so wa e using ull con ocal z-s acks (a ound 20) o each cell. Z-s acks
we e p e iously impo ed o Fiji so wa e o backg ound adjus men s and hen
decon ol ed using he blind-decon olu ion wiza d o Huygens so wa e.
S a is ical analysis
Fo mouse expe imen s, no speci ic blinding me hod was used bu mice in each sample
g oup we e selec ed andomly. The sample size (n) o each expe imen al g oup is
desc ibed in each co esponding igu e legend, and all expe imen s we e epea ed a leas
wi h h ee biological eplica es. G aphPad P ism so wa e was used o all s a is ical
analyses. Quan i a i e da a displayed as his og ams a e exp essed as means ± s anda d
e o o he mean ( ep esen ed as e o ba s). Resul s om each g oup we e a e aged
and used o calcula e desc ip i e s a is ics. Mann–Whi ney U- es (independen samples,
wo-sided) was used o pai wise compa isons among g oups a each ime poin .
S a is ical signi icance was se a a P alue <0.05.
22
42 Masie o, E. e al. Au ophagy is equi ed o main ain muscle mass. Cell Me ab 10,
507-515, (2009).
43 Suel es, M. e al. uPA de iciency exace ba es muscula dys ophy in MDX mice.
The Jou nal o cell biology 178, 1039-1051, (2007).
44 B ei k eu z, B. J., Jo gensen, P., B ei k eu z, A. & Tye s, M. AFM 4.0: a oolbox
o DNA mic oa ay analysis. Genome biology 2, SOFTWARE0001 (2001).
45 Hulsen, T., de Vlieg, J. & Alkema, W. BioVenn - a web applica ion o he
compa ison and isualiza ion o biological lis s using a ea-p opo ional Venn
diag ams. BMC Genomics 9, 488, (2008).
46 Sacco, A. e al. Sho elome es and s em cell exhaus ion model Duchenne
muscula dys ophy in mdx/mTR mice. Cell 143, 1059-1071, (2010).
47 C ews, L. e al. Selec i e molecula al e a ions in he au ophagy pa hway in
pa ien s wi h Lewy body disease and in models o alpha-synucleinopa hy. PLoS
One 5, e9313, (2010).
48 Pe digue o, E. e al. Gene ic analysis o p38 MAP kinases in myogenesis:
undamen al ole o p38alpha in ab oga ing myoblas p oli e a ion. The EMBO
jou nal 7, 1245-1256. (2007).
49 Kamen sky, L. e al. Imp o ed s uc u e, unc ion and compa ibili y o CellP o ile :
modula high- h oughpu image analysis so wa e. Bioin o ma ics 27, 1179-1180,
(2011).
50 Bol e, S. & Co delie es, F. P. A guided ou in o subcellula colocaliza ion analysis
in ligh mic oscopy. Jou nal o mic oscopy 224, 213-232, (2006).
23
EXTENDED DATA
Ex ended Da a 1. The educed au ophagy lux in quiescen sa elli e cells can be
pha macologically e ueled in i o.
a, Venn diag ams o o e lapping genes be ween a p o eos asis gene se (See
Supplemen a y Table 1) and genes signi ican ly up egula ed in quiescen sa elli e cells
om he indica ed publica ions o om ou gene exp ession mic oa ay da a compa ing
eshly FACS isola ed sa elli e cells om es ing muscle, o muscle inju ed wi h ca dio oxin
(CTX) o 72 h, om young WT mice. b, K-means clus e ing analysis (pe o med wi h
Gene-E, B oad Ins i u e) o he gene exp ession o he au ophagy- ela ed genes du ing
aging. Clus e s a e shown wi h hea maps o he no malized aw da a. Each column
ep esen s a di e en sample and each ow a di e en gene p obe. Red, inc eased
exp ession; whi e, neu al exp ession; blue, dec eased exp ession. c, Rep esen a i e
example o he FACS s a egy and ga ing scheme o isola e sa elli e cells om mice in
es ing condi ions. d, Pax7 and GFP immunos aining o eshly isola ed sa elli e cells om
es ing muscles o young and old GFP-LC3 mice. Scale ba , 5 μm. e, Elec on mic oscopy
images o young and old sa elli e cells on sec ions o es ing ibialis an e io (TA) muscle
o WT mice. A owheads indica e au ophagic esicles. Scale ba , 1 and 0.5 μm ( igh and
le , espec i ely). , Pax7 and GFP immunos aining on issue sec ions om es ing TA
muscles o young and old GFP-LC3 mice. A owheads indica e au ophagic esicles. Scale
ba , 5 μm. g, p62/ubiqui in (Ub) MFI. A owheads: p62/Ub-agg ega es colocaliza ion. h,
LC3 Wes e n blo o eshly isola ed sa elli e cells om young and old WT mice, ea ed
wi h Ba ilomycin (+Ba ) o ehicle o 4 h p io o collec ion. G aph shows LC3II
quan i ica ion, a e no maliza ion wi h Tubulin le els. i, p62 Wes e n blo ing o eshly
isola ed sa elli e cells om young and old WT mice. G aph shows p62 quan i ica ion, a e
no maliza ion wi h Tubulin le els. j, Quiescen sa elli e cells we e eshly isola ed om old
WT mice subjec ed o 2-week Rapamycin, Spe midine o ehicle (con ol) ea men . Cells
we e ea ed o no wi h Ba ilomycin 4 h p io analysis by immunos aining o LC3 ma ke .
Z p ojec ions o ep esen a i e luo escence mic oscopy images a e shown. Scale ba , 5
μm. k, Rep esen a i e luo escen mic oscopy images om Fig. 1d. Scale ba , 5 μm. Da a
show mean ± s.e.m. Compa isons by wo-side Mann-Whi ney U- es . P alues a e
indica ed. Numbe o samples, a,b) n=3 animals/g oup; g) n=35 (Young) and 66 (Old), 3
animals; h,i) n=3 animals/g oup.
Ex ended Da a 2. Reinduc ion o au ophagy escues p oli e a ion and educes
senescence in ge ia ic sa elli e, hus es o ing egene a i e capaci y.
a, T ansplan ed muscles om Fig. 2c we e immunos ained o GFP and o
Ki67/Pax7/MyoD/Mgn ( o deciphe he dis inc possible myogenic s a es o sa elli e cells in
he egene a ing muscle). Scale ba , 50 μm. b, Au ophagy lux analyzed by low cy ome y
in eshly isola ed sa elli e cells om es ing muscle o GFP-LC3 mice, ea ed o 48 h
wi h Rapamycin o ehicle (con ol). Sa elli e cells we e ea ed wi h Ba ilomycin (+Ba ) o
ehicle o 4 h p io o analysis. Resul s a e exp essed as a ela i e a ia ion o GFP–LC3
MFI in -Ba compa ed o +Ba condi ions. c, Wes e n blo analysis o phospho-S6 (pS6)
p o ein le els in young and ge ia ic sa elli e cells om WT mice, ea ed o 48 h wi h
Rapamycin o ehicle (con ol). G aph shows pS6 quan i ica ion, no malized o Tubulin. d,
As in Fig. 2c, pe cen age H2AX+/GFP+ o p16INK4a+/GFP+ cells om o al-GFP+ cells a e
quan i ied. Scale ba , 10 μm. e, Quan i ica ion B dU+ and SA-β-gal+ sa elli e cells, p e-
ea ed as in Fig. 2c and analysed a e 96h. , Quan i ica ion o senescen (SA-β-gal+)
24
sa elli e cells, isola ed om young and ge ia ic WT mice, p e- ea ed o 48 h wi h
Spe midine o ehicle (con ol) and cul u ed o 96 h. g, Quan i a i e eal- ime PCR (RT-
qPCR) analysis o A g7 exp ession on sa elli e cells in ec ed wi h LV-A g7 o LV-con ol
(LV-Co), and cul u ed o 96 h. h, GFP-LC3 sa elli e cells we e in ec ed wi h LV-A g7 o
LV-Co and ea ed wi h Ba ilomycin (+Ba ) o ehicle o 4 h p io o analysis. Au ophagy
lux was analyzed by low cy ome y and ep esen ed as in (b). Rep esen a i e images a e
shown. Scale ba , 10 μm. i, Muscle egene a ion expe imen by sa elli e cell
ansplan a ion: Equal numbe o sa elli e cells om young and ge ia ic mice in ec ed wi h
a len i i us o e exp essing A g7 gene (LV-A g7) o a len i i us con ol (LV-Co), which also
exp essed GFP, we e ansplan ed in o inju ed muscle o young immunode icien mice,
and collec ed 28 days la e . GFP exp ession in muscles was analyzed by immunos aining.
Quan i ica ion o GFP+ cells ( ibe s) pe muscle ield s. ansplan ed con ol- ea ed
sa elli e cells and ep esen a i e images a e shown. Scale ba , 75 μm. j, EDL ge ia ic
muscles, in ec ed wi h LV-A g7 o LV-Co, and g a ed on ecipien mouse-muscle, and
egene a ion analyzed 8-days-la e . F equency dis ibu ion o egene a ing ibe s by size.
Scale ba , 25 μm. Da a as mean ± s.e.m. Compa isons by wo-side Mann-Whi ney U- es .
P alues a e indica ed. Numbe o samples, b) n=20.000 cells/animal, 3 animals; c) n=3
animals/g oup; d) n=5 eng a men s/g oup; e-g) n=3 animals/g oup; h) n=20.000
cells/animal, 3 animals; i) n=3 eng a men s/g oup; j) n=4 eng a men s/g oup.
Ex ended Da a 3. Gene ic impai men o au ophagy in young quiescen sa elli e
cells leads o p ema u e senescence and impai ed muscle egene a ion.
a, RT–qPCR analysis o A g7 exp ession and Wes e n blo analysis o LC3, p62 and
Tubulin o sa elli e cells isola ed om A g7WT and A g7ΔPax7 mice. G aph shows he
quan i ica ion o p62 no malized o Tubulin. b, Quiescen sa elli e cells we e eshly
isola ed om A g7WT and A g7ΔPax7 mice which had been subjec ed o 2-week Rapamycin
o ehicle (con ol) ea men in i o. Cells we e ea ed o no wi h Ba ilomycin 4 h p io
analysis by luo escence mic oscopy. Z p ojec ions o ep esen a i e luo escence
mic oscopy images a e shown. Scale ba , 5 μm. c, Quan i ica ion o sa elli e cells in
es ing muscle o 3 mon h-old A g7WT and A g7ΔPax7 mice by low cy ome y analysis
(α7in eg in+
CD34+ cells pe g am o muscle issue). d, Rep esen a i e luo escen
mic oscopy images om Fig. 3d. Scale ba , 10 μm. e, RT-qPCR analysis o MyoD,
Myogenin (Mgn), and Ki67 exp ession in eshly isola ed quiescen sa elli e cells om
es ing muscle o A g7WT and A g7ΔPax7ER mice, 7 days a e amoxi en ea men . ,
Pe cen age o ac i a ed-sa elli e cells (Pax7+/MyoD+) om o al-Pax7+ cells (FACS-
isola ed 14-h pos -inju y om (a)). Scale ba , 50 μm. g, pS6/Lamp1-immunos aining o
cells om (a). Scale ba , 10 μm. h, H2AX p o ein le els pe nucleus on Pax7+ sa elli e
cells in TA muscles o A g7WT and A g7ΔPax7ER mice, 15 days pos -inju y. Rep esen a i e
images a e shown. Scale ba , 25 μm. i, Pax7+
sa elli e cells we e quan i ied ollowing
immunos aining on egene a ing muscles o A g7WT and A g7ΔPax7ER mice 7 and 15 days
pos ca dio oxin (CTX) inju y. j, Rep esen a i e images o hema oxilin/eosin s aining o
muscles a 7 days pos -inju y on muscles o A g7WT and A g7ΔPax7ER mice. Fibe size o
cen al-nuclea ed myo ibe s a 7 and 28 days pos -inju y is quan i ied. Scale ba , 50 μm.
k, TA muscles o A g7WT and A g7ΔPax7 mice we e inju ed by CTX injec ion and, 21 days
a e , hese muscles we e einju ed and analyzed 21 days la e (21+21 days pos -inju y).
The size o cen al-nuclea ed myo ibe s was quan i ied. Rep esen a i e images a e
shown. Scale ba , 50 μm. l, Pax7+/Ki67+
sa elli e cells we e quan i ied ollowing
immunos aining on egene a ing muscles o A g7WT and A g7ΔPax7ER mice 7 days pos
25
ca dio oxin (CTX) inju y. m, Equal numbe o quiescen sa elli e cells om A g7WT:GFP-
LC3 and A g7ΔPax7:GFP-LC3 mice (2-weeks -/+Rapamycin-p e- ea ed), ansplan ed as in
Fig. 2c, and immunos ained wi h indica ed-an ibodies 4-days-la e . Quan i ica ion
GFP+cells/muscle ield s. ansplan ed young WT sa elli e cells. Rep esen a i e images
a e shown. Scale ba , 75 μm. n, Pe cen age o GFP+/Ki67+ om o al GFP+ cells in
muscles om (m). o, Quan i ica ion o p oli e a ing (B dU+) and senescen (SA-β-gal+)
sa elli e cells, isola ed om A g7WT and A g7ΔPax7, p e- ea ed o 48 h wi h Spe midine o
Rapamycin (o con ol ehicle) and cul u ed o 96 h. Da a show mean ± s.e.m.
Compa isons by wo-side Mann-Whi ney U- es . P alues a e indica ed. Numbe o
samples, a) n=3 animals/g oup; c) n=7 animals/g oup; e-l) n=3 animals/g oup; m,n) n=4
eng a men s/g oup; o) n=3 animals/g oup.
Ex ended Da a 4. Au ophagy loss in sa elli e cells causes dys unc ional mi ophagy
and mi ochond ia accumula ion, leading o inc eased ROS and senescence.
a, p62 and ubiqui in (Ub) immunos aining on eshly isola ed sa elli e cells om es ing
muscle o 3 mon h-old A g7WT and A g7ΔPax7ER mice, 1-mon h-a e amoxi en- ea men .
A owheads indica e colocaliza ion o p62 and Ub agg ega es. Rep esen a i e images a e
shown. Scale ba , 5 μm. b, TOM20 and Lamp1 immunos aining on quiescen sa elli e
cells isola ed om young and ge ia ic WT mice. Mice we e subjec ed o 2-week
Rapamycin, Spe midine o T olox (o ehicle) ea men p io o analysis. Colocaliza ion
was calcula ed as he a ea occupied by he immuno luo escence co-localizing s aining on
pic u es wi h espec o he o al cellula a ea. The Pea son’s coe icien ( ) was used o
analyze he co ela ion o he in ensi y alues o g een and ed pixels in dual-channel
images. Z p ojec ions o ep esen a i e luo escence mic oscopy images a e shown. Scale
ba , 5 μm. c, Mi ochond ia quan i ica ion by Mi oT acke in quiescen sa elli e cells o old
mice, ea ed wi h Rapamycin o ehicle o wo weeks. d, Mi ochond ia (Mi oT acke ) in
young/ge ia ic cells. Sa elli e cells, p e- ea ed wi h CCCP o 1h (see Me hods), and
-/+Rapamycin o 24h. Pe cen age o Mi oT acke MFI educ ion +/-Rapamycin. e,
Mi ochond ial memb ane po en ial (MP) analysis: Sa elli e cells we e eshly isola ed om
young WT mice and ea ed o 1h wi h CCCP o DMSO (Con ol). Memb ane po en ial
(TMRM MFI/Mi oT acke G een MFI a io) o cells was calcula ed by low cy ome y
analysis a 1h and 24h a e CCCP ea men (being 100% he MP alue o con ol sa elli e
cells). , Mi ochond ia con en was quan i ied by Mi oT acke s aining o sa elli e cells om
young and ge ia ic WT mice and ea ed wi h Rapamycin o ehicle (con ol) o 48 h. Z
p ojec ions o ep esen a i e luo escence mic oscopy images a e shown. Scale ba , 5
μm. g, Mi ochond ia and ROS de ec ion by Mi oT acke and CellROX s aining,
espec i ely. Colocaliza ion was calcula ed as in (b). Z p ojec ions o ep esen a i e
luo escence mic oscopy images a e shown. Scale ba , 5 μm. h, Rep esen a i e images
o eshly isola ed sa elli e cells om es ing muscle o 3 mon h-old A g7WT and A g7ΔPax7
mice s ained wi h CellROX luo escen dye and p16INK4a an ibody. Scale ba , 5 μm. Da a
show mean ± s.e.m. Compa isons by wo-side Mann-Whi ney U- es . P alues a e
indica ed. Numbe o samples, a) n=36 (A g7WT) and n=38 (A g7ΔPax7ER), 3 animals;
b) n=23 (Young), 24 (Con ol), 42 (Rapamycin); 28 (Spe midine) and 21 (T olox), 3
animals; c) n=20000 cells/animal, 3 animals; d) n=10000 cells/animal, 4 animals; e, )
n=10000 cells/animal, 3 animals; g) n=18 (Young), 21 (Con ol), 15 (Rapamycin) and 13
(T olox), 3 animals.
Ex ended Da a 5. ROS in au ophagy-impai ed aged and A g7-null sa elli e cells
p<0.05
a
Pax7+ cells/100 Fibe s
A g7WT
A g7Pa
0
2
4
6
8
10
p<0.05
B du+ cells (%)
A g7WT
A g7Pa
0
10
20
30
40
50 p<0.003
A g7WT
A g7Pa
SA-ß-gal+ cells (%)
0
2
4
6
8
10
d
e
Figu e 3
0
2
4
6
8
Pax7+ cells/100 Fibe s
A g7WT
A g7Pa
p<0.008
bTmx
7 days 30 days
A g7WT A g7∆Pa
Analysis
0
20
40
60
%2+ cells
A g7WT
A g7Pa
p<0.02
pS6
S6
Tubulin
2
0.0
0.5
1.0
1.5
A g7WT
A g7Pa
2b a
p<0.05
0
100
200
300
400
500p<0.05
p66 a
A g7WT A g7Pa
Tubulin
g
cp16INK4a p21CIP1
p<0.04 p<0.04
p15INK4b
A g7WT
A g7Pa
a p
p<0.05
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
2.5
0.0
0.5
1.0
1.5
2.0
Young Old
a
0
p<0.01
Lamp1 MFI
Young
Old
2
4
6
Mi oT acke
0
2000
4000
6000
8000
p<0.04
LysoT acke
LysoT acke MFI
Young Old
Mi oT acke
A g7WT A g7 A g7WT A g7
Mi oT acke
p<0.04
Mi oT acke MFI
A g7WT
A g7
0
20000
40000
60000
LysoT acke
A g7WT
A g7
p<0.05
LysoT acke MFI
A g7WT
A g7
0
5000
10000
15000
20000
A g7WT
A g7
b
Figu e 4
Young Old
p<0.0001
2
4
6
8
0
Lamp1 MFI
Dapi
Lamp1
Dapi
Mi oT acke
Dapi
Lamp1
Dapi
Memb ane po en ial (%)
Memb ane po en ial (%)
Young
Old
0
50
100
150p<0.05
0
50
100
150p<0.05
Young
Old
Mi oT acke MFI
0
20000
40000
60000
80000p<0.02
e
p<0.04 p<0.0002
mRNA Rela i e exp ession
p16INK4a p21CIP1
b
0
10
20
30
40
50 p<0.04
0
5
10
15 p<0.04
B du+ cells (%)
SA-ß-gal+ cells (%)
% GFP+ cells/ sec ion
p<0.02
a
c
Figu e 5
0
100
200
300
400
500
T olox
Con ol
Ge ia ic
T olox
Con ol
Ge ia ic
0.0
0.5
1.0
1.5
0.0
0.5
1.0
1.5
T olox Con ol
GFP Dapi Me ge
0.0
0.2
0.4
0.6
0.8
1.0 p<0.02
p<0.03
p16INK4a/Tubulin Ra io
0.0
0.5
1.0
1.5
pS6/Tubulin Ra io
p<0.03
p<0.03
Con ol
Young
Ge ia ic
T olox
d
Tubulin
pS6
p16INK4a
Young Con ol T olox
Ge ia ic
p<0.04
CellROX MFI
Young
Old
0
200
400
600
800
Young
Old
Young Old
CellROX
Mi oT acke
Young
+ Ba
∆ Ma MFI (±Ba )
0
5000
10000
15000p<0.05
p<0.02
0
2000
4000
6000
8000
∆ C MFI (±Ba )
Young
Ge ia ic
A g7Pa
p<0.03
NS
c
Figu e 6
pS6/Tubulin Ra io
T olox
Con ol A g7∆Pa
p16INK4a/Tubulin Ra io
p16INK4a
pS6
S6
Tubulin
A g7WT
A g7∆Pa
Con ol T olox
0.0
0.5
1.0
1.5
p<0.05
p<0.05
0
1
2
3A g7WT
p<0.05
a
H2Aub ela i e
en ichmen (bound/inpu )
T olox
Con ol
Ge ia ic b
p<0.041
0
5
10 p<0.002
0
1
2
p<0.002
0
1
2
Exon 1aRD Exon 2
INK4a //
1.5
0.5
1.5
0.5
p<0.001 p<0.009
0
1
2
3
p<0.002 p<0.005
0
5
10 p<0.009
p<0.018
0
1
2
3
4
5
Exon 1aRD Exon 2
INK4a //
H2Aub ela i e
en ichmen (bound/inpu )
T olox
Con ol A g7∆Pa
A g7WT
Lin- (Sca1-, CD31-, CD11b- cells)
α7-in eg in
CD34
ba
c
LC3I
LC3II
Tubulin
Young Old
Ba +- + + +- - -
Young Old Young
Old
d
LC3II/Tubulin Ra io (±Ba )
p<0.05
0
1
2
3
4
p62
Tubulin
Young
Old p<0.03
p62/Tubulin Ra io
Young
Old
0
5
10
15
gh
Pax7
GFP-LC3
Dapi Me ge
GFP-LC3Pax7
Young Old
Me geDapi
Young
Old
Young Old e
Ex ended Da a 1
-
+ Ba + Ba + Ba + Ba
RapamycinCon ol Spe midineCon ol
Quiescence genes implica ed in p o eos asis: 236
1185 444
48
P o eos asis
Fukada e al
1186 481
47
P o eos asis
Palla acchina e al
1155 714
78
P o eos asis
Liu e al
1064 1820
169
P o eos asis
This s udy
Liu
(F eshly isola ed WT s
eshly isola ed BaCl2 inju ed)
Me hod: VCAM1+
CD31-/CD45-/Sca1-
Fukada e al
(F eshly isola ed WT s cul u ed)
Me hod: SM/C-2.6+ CD45-
Palla acchina e al
(F eshly isola ed WT s
eshly isola ed mdx)
Me hod: Pax3GFP+
This s udy
(F eshly isola ed WT s
eshly isola ed CTX inju ed)
Me hod: 7in eg in+CD34+
CD31-/CD45-/Sca1-
0 40 80 120 160
Au ophagy
Ubiqui in-p o easome
An ioxidan - esponse
Hea shock esponse
UPR
Numbe o genes
Young
Old
min max
Clus e 0
Young
Old
Young
Old
Clus e 1
Clus e 2
K means clus e ing analysis o gene exp es-
sion o au ophagy- ela ed genes (621) in old
cells
Clus e 2: Genes wi h educed exp ession in
old cells (386)
Signi ican ly down egula ed genes in old cells
belonging o Clus e 2 (236)
i
Old
LC3 Dapi LC3 Dapi
j
p62 Ub Dapi Me ge
Con olRapamycin
GFP-LC3
Ub
Young Old
p62 Me geDapi
0
p<0.0001
p<0.01
p62 MFI
Ub MFI
2
4
6Young
Old
Young
Old
0
2
4
6
8
k
Ge ia ic
Rapamycin
Con ol
pS6
Tubulin
Rapamycin
Con ol
pS6/Tubulin Ra io
Ge ia ic
0
2
4
6
8
10
Ex ended Da a 2
d
g
LV-CoLV-A g7
p<0.05
0
1
2
4
5
LV-A g7
LV-Co
p<0.05
0
50
100
150
A g7
0
50
100
150
200
mRNA Rela i e exp ession
GFP-LC3
Dapi
0
2
4
6
8
10
LV-A g7
LV-Co
SA-ß-gal+
Spe midine
Con ol
Spe midine
Ge ia ic
Con ol Young
h
p<0.05
p<0.001
Rapamycin
Con ol
Ge ia ic
+
LV-Co
LV-A g7
Ge ia ic
LV-A g7
GFP Dapi
p<0.05
0
200
400
600
800
Ge ia ic
NS
GFP DapiPax7
MyoD
Ki67
Mgn GFP Dapi
Me ge
Me ge
4 days ansplan
LV-Co
a b c
25m
GFP
Dapi Me ge
GFP Dapi Me ge
p16INK4a
γH2AX
Ge ia ic
Con olRapamycin
Ge ia ic
Con olRapamycin
ij
0
100
200
300
400
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
0
5
10
15
20
25
LV-Co
LV-A g7
Fibe size (µm2)
Fibe size (µm2)
p<0.03 Ge ia ic
LV-Co o LV-A g7
In ec ion
Ge ia ic WT mice
(dono )
Young mice
(hos )
Muscle g a ing
EDL muscle
Ge ia ic
LV-Co LV-A g7
Rela i e equency (%)
0
5
10
15
20
0
20
40
60
Con ol
Rapamycin
Ge ia ic
% p16INK4a+/GFP+ cells
2AX+/GFP+ cells
p<0.03
p<0.05
B dU+ cells (%)
SA-ß-gal+ cells (%)
e
0
2
4
6
8
10
Young
Con ol
Rapamycin Ge ia ic
LV-A g7
p<0.001
p<0.001
p<0.001
0
10
20
30
40 p<0.005
p<0.05
p<0.05
eMHC
d
Pax7 Me geDapi
A g7WT
A g7
2
4
6
8
A g7WT
A g7
e
c
g
A g7WT
A g7
5
1
1 5
5
1
1 5
5
1
1 5
2
A g7WT
A g7
A g7WT
A g7
+
b
aA g7WT A g7
A g7WT
A g7
5
1
15
2
25
5
1
1 5
A g7WT
A g7
A g7WT
A g7
++
A g7
2
4
6
+
A g7WT
A g7
2
4
6
8
1
+ Ba + Ba
A g7WT
A g7
LC3 Dapi + Ba + Ba
7+1
1
2
3
4
5
7 15
A g7WT
A g7
A g7WT
A g7
2
h
A g7WT A g7 A g7WT A g7
A g7WT
A g7
7 28
i
2
A g7WT
A g7Pax7
Pax7γH2AX Dapi Me ge
pS6 Lamp1 Me ge
A g7WT
A g7Pax7
Dapi
0
5
10
15
p<0.03
A g7WT
A g7Pax7ER
Pax7+/Ki67+ cells/ 100 Fibe s
7 days pos -inju y
0
5
10
15
20 A g7WT
A g7
GFP+/Ki67+ cells
p<0.04
4 days pos - ansplan a ion
j k
l m
o
n
GFP MyoD Mgn Dapi
A g7WT Con ol
A g7Pax7Con ol
A g7WT Rapamycin
A g7Pax7 Rapamycin
GFP+ cells/ sec ion
0
50
100
150p<0.05
NS
NS
GFP
A g7WT
A g7Pax7
Con ol Rapamycin
MyoD Mgn Dapi
A g7WT:GFP-LC3
A g7Pax7: GFP-LC3
Cell T ansplan a ion
4d Analysis
SCID mice
14h pos -inju y
Pax7 MyoD Dapi
p<0.04
Pax7+/MyoD+ cells
A g7WT
A g7Pax7
0
20
40
60
A g7WT
A g7Pax7
a b
c
d
g
CellROX p16INK4a Dapi Me ge
A g7WT
A g7
0
50
100
150
200
-100
0
100
200
300
Mi oT acke
h
Mi oT acke
CellRox
Dapi Me geMi oT acke CellRox
TYoung
0
20
40
60
80
Young
p<0.0001
Young
e
-60
-40
-20
0
20
40
(Mi oT acke )
Young
Ge ia ic
Young
Ge ia ic
Con ol
NS
p<0.05
p<0.05
( educ ion in o al
Analysis
Mi oT acke
Con ol
Mi oT acke MFI
Con ol
Old
0
500
1000
1500
2000p<0.04
YoungCon ol
Lamp1 TOM20
0
20
40
60
% o colocaliza ion (Lamp1/TOM20)
Young
Con ol
Spe midine
Ge ia ic
0.0
0.2
0.4
0.6
0.8
p<0.005 p<0.0001
p<0.0001
p<0.0001
p<0.0001 p<0.0001
p<0.0001
p<0.0001
Dapi Me ge
Ge ia ic
T olox
Ub p62 Me geDapi
A g7WT
A g7
p<0.0005
Ub MFI
8
6
4
2
0
p<0.002
A g7WT
A g7
p62 MFI
0
6
9
12
3
% o colocaliza ion
(Mi oT acke /CellROX)
Ba +- + + +- - -
T olox - + + +
+ + +
- - - + + ++
LC3I
LC3II
Tubulin 0
1
2
3
4
5
LC3II/Tubulin Ra io (±Ba )
T olox
Con ol
p<0.03
T olox
Con ol
0
50
100
150
200
250
Con ol
T olox
ROS
p<0.05 Ge ia ic
MFI (Ge ia ic s Young)
c d
e
h
g
Ex ended Da a 5
Ge ia ic
T olox Con ol
p62 Ub Dapi Me ge
GFP-LC3
p<0.002
0
1
2
3
4p<0.001
0
0.5
1
1.5
2
T olox
Con ol
Ub MFI
p62 MFI
Old
0
5
10
15
20
25
0
10
20
30
40
50
A g7WT LV-sh Sc amble
A g7Pax7LV-sh Sc amble
B du+ cells ()
SA--gal+ cells ()
A g7WT LV-sh p16INK4a
A g7Pax7 LV-sh p16INK4a
p<0.006
p<0.0001
p<0.03
p<0.04
0
2
4
6
8
10
SA--gal+ cells ()
p<0.03
p0.0007
0
20
40
60
80
p<0.05
p<0.05
B du+ cells ()
A g7WT Con ol
A g7Pax7Con ol
A g7WT T olox
A g7Pax7 T olox
p<0.01
0
5
p<0.003
0
1
2
3p<0.008
0
1
2
3
4
Exon 1aRD Exon 2
INK4a //
10
2Aub ela i e
en ichmen (bound/inpu )
Ge ia ic
Young
i
Ge ia ic
Young
+Ba
T olox
0
20
40
60
80
100
0
20
40
60
80
100
+ Ba + Ba
+ Ba + Ba
T olox
+Ba
T oloxCon ol
YoungGe ia ic
p<0.0001 p<0.0001
NS p<0.0001
RFP+ GFP+ punc a
RFP+ GFP+ punc a
Con ol Con ol
MgnMyoD GFP Dapi Me ge
Con olT olox
Ge ia ic
j k
Mi oT acke
0
500
1000
1500
2000
Mi oT acke MFI
p<0.05
a
CellROX
A g7WT A g7Pax7
p<0.04
A g7WT
A g7Pax7
A g7WT
A g7Pax7
0
5000
10000
15000
CellRX MFI
53BP1
Tubulin
A g7WT A g7Pax7
0
200
400
600
800
1000p<0.02
53BP1/Tubulin Ra io
A g7WT
A g7Pax7
Tubulin
Pa kin
0.0
0.1
0.2
0.3
0.4 p<0.05
Pa kin/Tubulin Ra io
b
T olox
GFP-LC3
Con ol
+Ba +Ba
T olox
Con ol
Old
0
50
100
150
200
GFP-LC3 MFI (±Ba )
p<0.05
Young Ge ia ic
c
Young Ge ia ic
H/E
e
YoungGe ia ic
Ex ended Da a 6
Human CD56 p16INK4a Dapi Me ge
p<0.02
15
20
25
30
35
40
45
B dU+ cells (%)
Young
Ge ia ic
d
pS6 Dapi
Young Ge ia ic
Con olRapamycin
Human
0
2
4
6
pS6
Tubulin
S6
Young
Ge ia ic
Ge ia ic
Ge ia ic
Ge ia ic
Con ol Rapamycin
Ge ia ic
Ge ia ic
Young Con ol
Rapamycin
pS6/Tubulin Ra io
p<0.03
p<0.03
g
l
Quiescence Senescence
Au ophagy / Mi ophagy
Aging
↑p16INK4a
Accumula ion o damaged o ganelles & mi ochond ia
↑ROS INK4a locus
de ep ession
p62
Tubulin
Young
Ge ia ic
Ge ia ic
Young
Ge ia ic
Ge ia ic
RapamycinCon ol
Mi oT acke
Young Ge ia ic
CellROX
Young Ge ia ic
-100
0
100
200
CellROX
0
500
1000
1500
Mi oT acke
Con ol
Rapamycin
MFI (Ge ia ic s Young) MFI (Ge ia ic s Young)
p<0.05
p<0.04
Young
Ge ia ic
Mi oT acke
Con olRapamycin
p<0.001
p<0.001
SA-ß-gal+ cells (%)
Rapamycin
Con ol
Young
Ge ia ic
0
20
40
60
RapamycinCon ol
h
Con ol
Rapamycin
Rapamycin
Con ol
Human
0.0
0.5
1.0
1.5
2.0 Young
Ge ia ic
Ge ia ic
Con ol
Rapamycin
p62/Tubulin Ra io
p<0.03
p<0.03
i
Young Ge ia ic
j
CellROX
k
Young
Human
a
pS6/Tubulin Ra io
LV-sh Sc amble
LV-sh p16INK4a
A g7Pax7
p16INK4a/Tubulin Ra io
0.0
0.2
0.4
0.6
0.8 p<0.05
0.0
0.5
1.0
1.5 p<0.05
Tubulin
S6
pS6
A g7∆Pax7
LV-sc amble LV-shp16
p16INK4a
0
50
100
150
200
p<0.0006
Fibe sie (m2)
LV-sh Sc amble
LV-sh p16INK4a
A g7Pax7
LV-sh Sc amble LV-sh p16INK4a
b
HE
eMHC
