summary of the c. elegans lifespan experiments all rnai feeding conditions and different treatments...

Supplementary information

Supplementary Table 1 Summary of the C. elegans lifespan experiments

Cumulative statistics Statistics of individual expts.

Stra

in

Bac

teria

/RN

Ai

Com

poun

d

No.

of e

xpts

.

Mea

n lif

espa

n [d

ays]

(tr

eatm

ent/v

eh)

chan

ge in

lif

espa

n [%

]

S.E.

M.

(trea

tmen

t/veh

)

No.

of a

nim

als

(trea

tmen

t/veh

)

Mea

n lif

espa

n (d

ays)

(tr

eatm

ent/v

eh)

chan

ge in

lif

espa

n [%

]

P-va

lue

No.

of a

nim

als

(trea

tmen

t/veh

)

N2 OP50 UA 50 µM 9 23.7/16.3 +45.4 0.30/0.23 447/536 26.4/17.8 +48.3 0.0001 50/37 29.2/17.9 +63.1 0.0001 50/54

26.8/16.7 +60.4 0.0001 44/47 24.2/18.3 +32.2 0.0001 53/76 20.7/13.6 +52.3 0.0001 64/64 22.3/15.3 +45.7 0.0001 68/83 18.3/14.7 +24.5 0.0001 54/75 21.8/14.2 +53.5 0.0001 36/52 24.1/18.9 +27.5 0.0002 28/48

N2 OP50 EA 50 µM 2 15.4/16.1 -4.3 0.49/0.53 112/112 14.3/13.6 +5.1 0.4 74/64 17.6/18.9 -6.8 0.3 38/48

N2 OP50 UB 50 µM 2 22.0/16.1 +36.6 0.58/0.53 86/112 21.8/13.6 +60.3 0.0001 50/64 22.3/18.9 +18.9 0.0049 36/48

N2 OP50 UC 50 µM 2 21.9/16.1 +36.0 0.64/0.53 99/112 21.1/13.6 +55.1 0.0001 72/64 23.2/18.9 +22.7 0.0013 27/48

N2 OP50 UD 50 µM 2 19.4/16.3 +19.0 0.58/0.49 140/136 17.4/13.6 +27.9 0.0002 68/64 21.3/18.3 +16.4 0.0072 68/76 N2 OP50 UA 10 µM* 2 22.7/18.3 +24.0 0.52/0.47 120/131 22.2/17.9 24.0 0.0007 53/49

23.0/18.5 24.3 0.0001 67/82 N2 OP50 UA 30 µM** 2 25.9/18.4 +40.7 0.60/0.49 108/129 26.1/18.3 42.6 0.0001 30/44

25.7/18.4 39.6 0.0001 78/85 N2 OP50 UA 50 µM

dev. only 2 17.6/16.7 +5.4 0.61/0.54 89/100 14.3/14.2 +0.7 0.9 51/52

21.1/18.9 +11.6 0.08 38/48 N2 OP50 UA 50 µM

ad. only 3 22.7/16.5 +37.5 0.39/0.38 167/218 24.2/14.2 +70.4 0.0001 36/52

22.7/16.0 +41.8 0.0001 71/90 21.6/18.3 +18.0 0.0037 60/76

N2 OP50 UA 50 µM until day 4

2 22.0/16.7 +31.7 0.56/0.51 118/128 19.2/14.2 +35.2 0.0001 44/52 23.4/18.3 +27.8 0.0001 74/76

N2 OP50 UA 50 µM until day 8

2 20.4/17.1 +19.3 0.58/0.44 138/166 19.4/16.0 +21.2 0.0001 72/90 21.4/18.3 +16.9 0.0026 66/76

N2 OP50 UA 50 µM the first 4 days

2 20.6/16.4 +25.6 0.47/0.40 161/169 20.8/16.0 +30.0 0.0001 75/90 20.3/16.8 +20.8 0.0001 86/79

N2 OP50 UVkilled

UA 50 µM ad. only

2 25.3/20.4 +24.0 0.45/0.56 119/129 24.3/19.7 +23.3 0.0001 43/51 25.6/21.0 +21.9 0.0001 76/78

daf-16 (mu86)

OP50 UA 50 µM 2 19.1/14.2 +34.5 0.38/0.40 121/113 17.2/13.6 +26.4 0.0001 72/71 21.8/15.3 +42.5 0.0001 49/42

eat-2 (ad465)

OP50 UA 50 µM 2 27.5/20.2 +36.1 0.39/0.62 76/106 29.5/20.1 +46.7 0.0001 50/66 33.2/21.9 +51.6 0.0001 26/40

aak-2 (ok524)

OP50 UA 50 µM 2 17.8/16.5 +7.8 0.41/0.34 109/121 17.4/16.4 +6.1 0.2 66/66 18.4/16.8 +9.5 0.003 43/55

mev-1 (kn1)

OP50 UA 50 µM 2 13.1/12.3 +6.5 0.41/0.35 112/138 13.1/11.8 +11.0 0.03 57/78 13.2/12.9 +2.3 0.7 55/60

N2 HT115 UA 50 µM 4 21.6/17.9 +20.6 0.42/0.36 217/233 23.1/18.3 +26.2 0.0001 57/68 24.8/19.8 +25.2 0.0001 47/50 18.9/16.0 +18.1 0.0005 77/65 20.6/18.0 +14.4 0.01 36/50

N2 bec-1 UA 50 µM 2 19.6/18.1 +8.3 0.39/0.42 103/112 19.8/18.5 +7.0 0.1 49/57 19.4/17.8 +8.9 0.08 54/55

N2 vps-34 UA 50 µM 2 16.0/15.2 +5.2 0.45/0.41 101/94 15.9/15.0 +6.0 0.4 56/50 16.5/15.7 +5.1 0.4 45/44

N2 pink-1 UA 50 µM 2 19.3/18.4 +4.9 0.53/0.52 105/102 21.7/19.4 +11.8 0.07 49/51 17.1/17.4 -1.7 0.6 56/51

Nature Medicine: doi:10.1038/nm.4132

Ryu et al. – Urolithin A induces mitophagy and improves muscle function

N2 dct-1 UA 50 µM 2 18.1/18.2 -0.5 0.38/0.39 108/105 17.7/17.4 +1.7 0.4 68/64 18.8/19.5 -3.6 0.1 40/41

N2 sqst-1 UA 50 µM 2 17.5/17.8 -1.7 0.33/0.38 109/106 16.9/17.4 -2.8 0.3 68/69 18.5/18.5 +0.0 0.6 41/37

N2 skn-1 UA 50 µM 2 12.4/13.4 -7.4 0.30/0.35 114/123 12.3/13.8 -10.8 0.0029 82/77 12.9/12.7 +1.5 0.9 32/46

N2 OP50 UA 50 µM +NAC

2 22.7/17.0 +33.5 0.43/0.49 152/165 23.3/19.0 +22.6 0.0005 76/74 22.1/15.3 +44.4 0.0001 76/91

N2 OP50 UA 50 µM +Para 4mM D1

2 5.6/4.9 +14.3 0.18/0.17 77/102 3.4/3.1 +9.6 0.02 31/38 6.6/5.9 +11.8 0.0032 46/64

N2 OP50 UA 50 µM +Para 4mM D8***

2 6.4/5.7 +12.3 0.10/0.15 45/70 6.4/5.9 +8.4 0.04 33/43 5.7/5.2 +9.6 0.03 12/27

Summary of mean lifespan and statistical analysis (P-values) for lifespan experiments

including all RNAi feeding conditions and different treatments displayed in Figures 1b—e, 2a,

3h, and Supplementary Figures 1c,d, 3b, 5b,c and e. veh corresponds to 1% DMSO in the

NGM medium, unless stated otherwise. *veh corresponds to 0.2% DMSO, **veh corresponds

to 0.6% DMSO, *** day 8 (D8) of adulthood is considered as day 0 of the lifespan assay. P-

values less than 0.05 are considered statistically significant, demonstrating that the lifespan

of the populations are different. Cumulative statistics and statistics of individual experiments

are shown for each conditions. The total number of individuals scored/censored is shown.



Supplementary Table 2 Summary of the C. elegans mobility experiments

Days Treatment N(trials)

1 vehicle 54(2) 1 EA 50 µM 66(2) 1 UA 50 µM 59(2) 1 UB 50 µM 71(2) 5 vehicle 60(2) 5 EA 50 µM 58(2) 5 UA 50 µM 58(2) 5 UB 50 µM 58(2) 8 vehicle 44(2) 8 EA 50 µM 52(2) 8 UA 50 µM 42(2) 8 UB 50 µM 47(2) 1 vehicle 30(1) 1 UC 50 µM 44(1) 1 UD 50 µM 36(1) 5 vehicle 30(1) 5 UC 50 µM 33(1) 5 UD 50 µM 36(1) 8 vehicle 31(1) 8 UC 50 µM 26(1) 8 UD 50 µM 30(1)

Summary of the number of worms and experiments for the mobility assays displayed in

Figure 1g and Supplementary Figure 2b.



Supplementary Table 3 List of the C. elegans strains

Strain name Genotype

N2 Wild-type Bristol CF1038 daf-16(mu86) I CL2070 dvIs70[hsp-16.2p::GFP+rol-6(su1006)] CL2166 dvIs19 [(pAF15)gst-4p::GFP::NLS] DA2123 adIs2122[lgg-1p::GFP::lgg-1+rol-6(su1006)] DA465 eat-2(ad465) II RB754 aak-2(ok524) X

RW1596 myo-3(st386)V;stEx30[myo-3p::GFP+rol-6(su1006)] SJ4100 zcIs13[hsp-6::GFP] SJ4005 zcIs4[hsp-4::GFP] SJ4143 zcIs17[ges-1::GFP(mit)] SJ4103 zcIs14[myo-3::GFP(mit)] TK22 mev-1(kn1) III

VC1024 pdr-1(gk448) III



Supplementary Table 4 List of primers used in C. elegans

Gene Symbol Gene ID Forward Reverse

act-1 179535 CTACGAACTTCCTGACGGACAAG CCGGCGGACTCCATACC

Y45F10D.4 178344 GTCGCTTCAAATCAGTTCAGC GTTCTTGTCAAGTGATCCGACA

cco-1 172832 GCTCGTCTTGCTGGAGATGATCGTT GGTCGGCGTCGACTCCCTTG

phi-37 173134 GCTCTTAACACCGCCGTCA CCATCTCCGACTGGAACGTC

mev-1 260040 ATCGATCGTCACCAAGTCCG GGAATCCGGAGAGCATCCAG

nduf-6 184827 CAACAAAGCCACCAACTCCG TATCCCCGCAATCCTCTGGA

sdha-1 181108 CTTGCACTGGAGACGGAACT TGCTCTGCTGGAAGATGGTG

mrps-5 179721 ACGTCGCCCACGTGGATTCG TGAAGACCTTTTCGTTCGGCCAGT

bec-1 177345 CTGTCAGCATCCGTTGAGGT AGAGCGTCAGAGCAATCATTACA

vps-34 172280 ATGATTCCAGGTATGCGGGC CTGACGAGCAAGTTGAGAGGA

rheb-1 176327 ACAAGACGGATCTCAGCACG TCGAACACCTCATGCACTCG

pink-1 173918 AAGCACCAGAAATTGCGACG ACGAGATGGGAGTGCTGGTA

sqst-1 178139 ATCCGCTCCTCACCAAATGC TGTTGGACGAAGGGGAACAG

dct-1 181053 GCAAAAGCCGTCTCAAACCC ACCCACGATTCTGACATACCA

skn-1 177343 ACAGTGCTTCTCTTCGGTAGC GAGACCCATTGGACGGTTGA

nd-1 2565698 AGCGTCATTTATTGGGAAGAAGAC AAGCTTGTGCTAATCCCATAAATGT

act-3 179533 TGCGACATTGATATCCGTAAGG GGTGGTTCTCCGGAAAGAA

MTCE.26 2565700 GGTTGTGGGACTAGGTGAACA CAGGGTGCCCCATTGTTCTT



Supplementary Table 5 List of primers used in C2C12 cells and mice

Gene Symbol Gene ID Forward Reverse

B2m 12010 TTCTGGTGCTTGTCTCACTG TATGTTCGGCTTCCCATTCT

Rplp1 56040 AGATTCGGGATATGCTGTTGG AAAGCCTGGAAGAAGGAGGTC

Actb 11461 GAGACCTTCAACACCCC GTGGTGGTGAAGCTGTAGCC

p62 18412 GCTGAAGGAAGCTGCCCTAT TTGGTCTGTAGGAGCCTGGT

Park2 50873 CCGAATCACCTGACGGTTCA TCTGGCTGCTTCTGAATCCC

Lc3b 67443 CACTGCTCTGTCTTGTGTAGGTTG TCGTTGTGCCTTTATTAGTGCATC

Atg8l 57436 GAGGACCACCCCTTCGAGTA GTGGGAGGGATGGTGTTGTT

Ulk1 22241 TCCCTACACACCTTCTCCCC AGCCAACAGGGTCAGCAAAT

Atg5 11793 GGAGAGAAGAGGAGCCAGGT GCTGGGGGACAATGCTAATA

Atg7 74244 GCCTAACACAGATGCTGCAA TGCTCTTAAACCGAGGCTGT

Becn1 56208 CCGCGGTAGAACGAGCC AAGTAATGGAGCTGTGAGTTCCT

Pik3c3 225326 GTGAAGTACCCTGACCTGCC AGTCATGCATTCCTTGGCGA

Lamp2 16784 TGTGCAACAAAGAGCAGGTG TCCAGTATGATGGCGCTTGAG

Atg12 67526 TAAACTGGTGGCCTCGGAAC ATCCCCATGCCTGGGATTTG

Myh1 17879 CGGTCGAAGTTGCATCCCTA TTCTGAGCCTCGATTCGCTC

Myh4 17884 GTCCTTCCTCAAACCCTTAAAGT AAAAGGCTTGTTCTGGGCCT

Myh6 17888 ATATAAAGGGGCTGGAGCACTG CTTTCGGAGGTACTGGGCTG

Myh2 17882 GAGGCTGACTCGTCCTGCT GCTCCGCCACAAAGACAGAT

Ucp2 22228 CTACAGATGTGGTAAAGGTCCGC GCAATGGTCTTGTAGGCTTCG

Hk2 15277 TCTGGCTCTGAGATCCATCTTCA CCGGCCTCTTAACCACATTCC

mt-Co2 17709 GTTGATAACCGAGTCGTTCTGC CCTGGGATGGCATCAGTTTT

mt-Rnr2 17725 CCGCAAGGGAAAGATGAAAGAC TCGTTTGGTTTCGGGGTTTC



Supplementary Table 6 List of antibodies

Antibody Supplier Reference # Validation

pAMPK-α (T172) Cell Signaling 2531 Validation is provided on the manufacturer’s website.

AMPK- α Cell Signaling 2603 Validation is provided on the manufacturer’s website.

LC3 A/B Cell Signaling 4108 Validation is provided on the manufacturer’s website.

Ubiquitin Cell Signaling 3933 Validation is provided on the manufacturer’s website.

p62/SQSTM1 Progen GP62-C The antibody was validated in articles belong to PMID: 19200883 and 23202295.

β-actin Sigma A5441 Validation is provided on the manufacturer’s website.

HSP90 BD Biosciences 610418 Validation is provided on the manufacturer’s website.

GAPDH Santa Cruz sc-25778 Validation is provided on the manufacturer’s website.

CRIF1 Santa Cruz sc-374122 Validation is provided on the manufacturer’s website.

Total OXPHOS WB Antibody Cocktail

Abcam ab110413 Validation is provided on the manufacturer’s website.

SDHA Abcam ab14715 Validation is provided on the manufacturer’s website.

VDAC1 Abcam ab14734 Validation is provided on the manufacturer’s website.

TOM20 Santa Cruz sc-11415 Validation is provided on the manufacturer’s website.

TOM40 Santa Cruz sc-11414 Validation is provided on the manufacturer’s website.

PINK1 Novus BC100-494 The antibody was validated in articles belong to PMID: 24784582, 18397367 and 18003639.



a

Time (d)

Sur

viva

l (%

)

vehicle, OP50UA, OP50

c

Time (d)

Sur

viva

l (%

)

vehicle, HT115UA, HT115

d

b

HT1

15 A

595

0

0.1

0.2

0.3

0.4

0.5

OP5

0 A 59

5

hours at 37°C0 1 2 3 4 5 6 7 8

DMSO 1%UA 50 μM

0

0.1

0.2

0.3

0.4

0.5

OP5

0 A 59

5

hours at 37°C0 1 2 3 4 5 6 7 8

DMSO 0.5%UA 25 μM

0

0.1

0.2

0.3

0.4

0.5

OP5

0 A 59

5

hours at 37°C0 1 2 3 4 5 6 7 8

0

0.1

0.2

0.3

0.4

hours at 37°C0 1 2 3 4 5 6 7 8

DMSO 2%UA 100 μM

DMSO 0.5%UA 25 μM

0

0.1

0.2

0.3

0.4

hours at 37°C0 1 2 3 4 5 6 7 8

HT1

15 A

595

DMSO 1%UA 50 μM

0

0.1

0.2

0.3

0.4

hours at 37°C0 1 2 3 4 5 6 7 8

HT1

15 A

595

DMSO 2%UA 100 μM

0 10 20 30 400

20

40

60

80

100

0 10 20 30 40 500

20

40

60

80

100

P < 0.0001 P < 0.0001

e

50 worms

OP50+

cpd 1OP50

+cpd 2

f

Supplementary Fig. 1

0

0.5

1.0

0

0.5

1.0

0

0.5

1.0

vehi

cle

vehi

cle

UA

UA

UA

vehi

cle

Frac

tion

of w

orm

s 2h

vehi

cle

vehi

cle

UA

UA

UA

vehi

cle

4h

Frac

tion

of w

orm

s

Frac

tion

of w

orm

s

vehi

cle

vehi

cle

UA

UA

UA

vehi

cle

6h

Supplementary Figure 1 Bacterial metabolism is not required for the UA-mediated longevity

phenotype. (a,b) Addition of UA slows the growth of bacterial strains OP50 (a, n = 5) and

HT115 (b, n = 5) at 25, 50 and 100 μM compared to the equivalent DMSO concentration. (c)

UA treatment extends lifespan of worms fed with OP50 by 45.4% (vehicle OP50, mean 16.3

± 0.2 days; UA OP50, mean 23.7 ± 0.3 days, P < 0.001). (d) UA treatment extends lifespan

of worms fed with HT115 by 20.6% (vehicle HT115, mean 17.9 ± 0.3 days; UA HT115, mean

21.6 ± 0.4 days, P < 0.001). (e,f) UA is not repulsive for the worms. (e) Scheme of the

bacterial avoidance assay. (f) Worms did not have any preference between UA and vehicle

as measured during the bacterial avoidance assay (n = 5). Values are mean ± s.e.m. Data

are representative of at least two independent experiments. UA was used at 50 µM unless



stated otherwise. Vehicle corresponds to 1% DMSO unless stated otherwise. See also

Supplementary Table 1.



050

100150200250

020406080

100

Day 7 Day 14

*** *

***

050

100150200250

***

020406080

100

P = 0.1

1 5 80

1

2

3

4

5

1 5 80

1

2

3

4

5

pum

ping

/min

*

050

100150200250

020406080

100

**

pum

ping

/min

pum

ping

/min

1 5 80

1

2

3

4

5

**

mob

ility

(A.U

.)

Time (d)

mob

ility

(A.U

.)

Time (d)

mob

ility

(A.U

.)

Time (d)

vehicleEA

vehicleUB

vehicleUC

a

b


1 5 80

1

2

3

4

5

mob

ility

(A.U

.)

vehicleUD

050

100150200250

**

020406080

100

pum

ping

/min

d

L1-L3 L4 Y.Ad

num

ber o

f pro

geny

vehicleUA

totalc

0

50

100

150

0

20

40

60

80

100

0

20

40

60

80

100

0

300

Ad

fract

ion

of w

orm

s(a

fter 4

8 h)

vehicleUA

1 2 3 4Time (d)

5L1-L3 L4 Y.Ad Ad

***

*

**

Time (d)

vehicleEA

vehicleUB

vehicleUC

vehicleUD

Day 7 Day 14 Day 7 Day 14 Day 7 Day 14fra

ctio

n of

wor

ms

(afte

r 72

h)

Supplementary Figure 2 Phenotypic effects of ellagic acid and other urolithins. (a,b) UB

increases pharyngeal pumping (a) and activity (b) similar to UA, while UC and UD, but not

EA, had mild to no benefit during aging. For a, graphs represent boxplot showing min. to max.

*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by unpaired t-test. For b, values are mean ± s.e.m. **P ≤

0.01; ***P ≤ 0.001 by two-way ANOVA followed by Bonferroni posttests. (c,d) UA has no

significant impact on development (c, n = 5) but slightly increases fertility (d, n = 5). Values

are mean ± s.e.m. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001 by two-way ANOVA followed by

Bonferroni posttests. Data are representative of at least two independent experiments. All

pharmacological treatments were made at 50 µM. Vehicle corresponds to 1% DMSO. See

also Supplementary Table 2.




b

0 10 20 30 400

20

40

60

80

100

0 10 20 30 400

20

40

60

80

100

0 10 20 30 400

20

40

60

80

100

Time (d)

Surv

ival (%

)

a

larval development adulthoodday 4day 0 day 8

12

534

vehicle

UA full life

UA specific timing

dc

AT

P c

onte

nt

(RL

U .

10

3)

Day 1

AT

P c

onte

nt

(RL

U .

10

3)

Day 8

e

0

1

2

3

4

vehic

le

UA

V1

IV2

SV

2

0

20

40

60

80

100 **

f

ve

hic

le

UA

37

°C (

1h

)

***

0

5

10

15

ve

hic

le

UA

mrp

s-5

Fold

induction

(hsp

-6::g

fp) ***

ve

hic

le

UA

Tu

nic

a. 0

2

4

6

8

ve

hic

le

UA

Acry

lam

ide

***

0

2

4

6

8

10

***

0

0.5

1.0

1.5

2.0

2.5

Surv

ival (%

)(1)

Surv

ival (%

)0 10 20 30 40

0

20

40

60

80

100

0 10 20 30 400

20

40

60

80

100

Surv

ival (%

)

Surv

ival (%

)(2) (3) (4) (5)

Time (d) Time (d) Time (d) Time (d)

n.s.

vehicleUA

vehicleUA

Fold

induction

(hsp

-16.

2::g

fp)

Fold

induction

(hsp

-4::g

fp)

Fold

induction

(gst

-4::g

fp)

Supplementary Figure 3 Timing requirement of UA for the lifespan extension and its effects

on mitochondrial function and stress resistance pathways in C. elegans. (a) Scheme showing

the different timing conditions of UA treatment. (b) UA treatment during the larval stage only

(from eggs until L4 larval stage) has no effect on lifespan (vehicle, mean 16.7 ± 0.5 days; UA

larval development only, mean 17.6 ± 0.6 days, P = 0.2 compared to vehicle) (1). UA

treatment during the adulthood only (from young adult stage until death) extends lifespan by

37.5% (vehicle, mean 16.5 ± 0.4 days; UA adulthood only, mean 22.7 ± 0.4 days, P < 0.001

compared to vehicle) (2). UA treatment from day 4 of adulthood extends lifespan by 31.7%

(vehicle, mean 16.7 ± 0.5 days; UA from day 4 of adulthood, mean 22.0 ± 0.5 days, P <

0.001 compared to vehicle) (3). UA treatment from day 8 of adulthood extends lifespan by

19.3% (vehicle, mean 17.1 ± 0.4 days; UA from day 8 of adulthood, mean 20.4 ± 0.6 days, P



< 0.001 compared to vehicle) (4). UA treatment during the 4 first days of adulthood extends

lifespan by 25.6% (vehicle, mean 16.4 ± 0.4 days; UA during the 4 first days of adulthood,

mean 20.6 ± 0.5 days, P < 0.001 compared to vehicle) (5). (c) BN-Page analysis in worms

reveals a reduced abundance of the mitochondrial complexes after 1 day of UA treatment.

S= super complexes; V2= dimeric complexes V; V1= complexes V; IV2= dimeric complexes IV.

(d,e) At Day 1 of adulthood, UA reduces ATP content (d, n = 4). At Day 8, ATP content is

equal in vehicleand UA treated worms (e, n = 3). Values are mean ± s.e.m. **P ≤ 0.01; n.s.

not significant by unpaired t-test. (f) UA had no effect on the mitochondrial stress (n = 9),

heat shock stress (n = 10), ER stress (n = 5) and oxidative stress (n = 12) as determined

using the hsp-6::GFP, hsp-16.2::GFP, hsp-4::GFP and gst-4::gfp reporters respectively. As

positive controls, mrps-5 RNAi, 1h at 37°C, exposure to tunicamycin (Tunica.) and

acrylamide were used to induce hsp-6::gfp, hsp-16.2::gfp, hsp-4::gfp and gst-4::gfp reporters,

respectively. Values are mean ± s.e.m. ***P ≤ 0.001 by one-way ANOVA. Data are

representative of at least two independent experiments. UA was used at 50 µM. Vehicle

corresponds to 1% DMSO. See also Supplementary Table 1.



a


0

5

10

15

0

5

10

15

0

5

10

15

0

1

2

3

4

0

1

2

3

4

0

1

2

3

4

mobili

ty (

A.U

.)

bec-1 pink-1vector only

mobili

ty (

A.U

.)

bec-1 pink-1vector only

***

*

*

f

0 1 2 3 4

bec-1

vps-34

rheb-1

pink-1

sqst-1

dct-1

skn-1

******

***

*****

**

vehicleUA

D1 bec-1

vps-34

rheb-1

pink-1

sqst-1

dct-1

skn-1

vehicleUA

D8

b

c

g

d e

0 0.5 1.0 1.5 2.0

*

P = 0.0005P < 0.0001

mRNA level

(fold induction)

mRNA level

(fold induction)

Fold

induction

(pm

yo-3m

tGF

P)

vehicleUA

ev dct-1

***

n.s.

Fold

induction

(pm

yo-3m

tGF

P)

vehicleUA

ev sqst-1

0

1

2

3

4

*

0

0.5

1.0

1.5

2.0

*

n.s.

OC

R

(pm

ole

s/m

in/w

orm

s)

0

10

20

30

40

0

10

20

30

40

0

5

10

15

20

25

0

5

10

15

20

25vehicle

UApink-1 RNAi, D1vector only, D1

basal FCCP basal FCCP

OC

R

(pm

ole

s/m

in/w

orm

s) pdr-1(gk448), D1wild-type, D1

vehicle

UA

**

basal FCCP basal FCCP

vehicle UA

***

Siz

e o

f m

ichondria (

A.U

.)p

ges-

1mtG

FP

h

vehicleUA

***

0

0.5

1.0

1.5

2.0

2.5

**

mR

NA

level

(fo

ld in

du

ctio

n)

fzo-1 opa-1

j

0

20

40

60

80

0

50

100

150

vehicle UA

***

i

vehicleUA

Day 1 Day 1 Day 1

vehicleUA

Day 8 Day 8 Day 8

Siz

e o

f m

ichondria (

A.U

.)p

myo

-3m

tGF

P

***

*

Supplementary Figure 4 Mitophagy is activated upon UA treatment in C. elegans. (a) UA

significantly increases the transcription of autophagy and mitophagy markers at day 1 (n = 5)

and 8 (n = 5) of adulthood. P < 0.0001 and 0.0005 corresponds to significance of the

treatment over the entire panel of genes after 2-way ANOVA test. *P ≤ 0.05; **P ≤ 0.01; ***P

≤ 0.001 corresponds to the significance of the Bonferroni posttest for specific genes. (b,c)

RNAi of sqst-1 (b, n = 8) and dct-1 (c, n = 8) suppresses the decrease in mitochondrial



content induced by UA in the muscle mtGFP reporter strain (pmyo-3mtGFP) at day 1 of

adulthood. Values are mean ± s.e.m. *P ≤ 0.05; ***P ≤ 0.001; n.s. not significant by unpaired

t-test. (d,e) RNAi of bec-1 and pink-1 does not impact mobility at day 1 of adulthood (d).

However, both genes are required for the improvement of mobility induced by UA observed

at day 8 of adulthood (e). Values are mean ± s.e.m. *P ≤ 0.05; ***P ≤ 0.001 by unpaired t-test.

(f,g) RNAi of pink-1 (f, n = 20) or mutation of pdr-1, the worm homolog of PARK2, (g, n = 20)

suppresses the decrease in basal OCR measured in UA treated worms. FCCP was used at

10 µM. Values are mean ± s.e.m. *P ≤ 0.05 by unpaired t-test.. (h,i) UA decreases the

average size of mitochondria in intestine (h, n = 5) and muscle (i, n = 5) mtGFP reporter

strains in worms at day 1 of adulthood. Values are mean ± s.e.m. ***P ≤ 0.001 by unpaired t-

test. (j) UA decreases the expression of the fusion genes fzo-1, the worm homolog of

mitofusin 2 (Mfn2), and opa-1 (n = 5). Values are mean ± s.e.m. **P ≤ 0.01; ***P ≤ 0.001 by

unpaired t-test. Data are representative of at least two independent experiments. UA was

used at 50 µM. Vehicle corresponds to 1% DMSO.



aSupplementary Fig. 5

c

0

20

40

60

80

100

P < 0.00010

20

40

60

80

100 vehicle, NACUA ad, NAC

P < 0.0001

e

0 10 20 30 40 0 10 20 30 40

Sur

viva

l (%

)

Time (d)

larval development adulthood

day 0 day 8 day 15

b

Treatment with vehicle or UAParaquat

Survival assay (b)

Treatment with vehicle or UAParaquat

Survival assay (c)

0

0.5

1.0

1.5

vehi

cle

UA

Fold

indu

ctio

n(M

itoS

ox)

n.s.

d

8 10 12 14 160

50

100

P = 0.0034

vehicle, D8UA, D8

0 2 4 6 80

50

100

Sur

viva

l (%

)(P

araq

uat 4

m M

)

P = 0.006

vehicle, D1UA, D1

Sur

viva

l (%

)(P

araq

uat 4

m M

)

Sur

viva

l (%

)

Time (d)

Time (d) Time (d)

vehicleUA ad

Supplementary Figure 5. UA prolongs lifespan independently of ROS. (a) Scheme showing

the different timing conditions of UA treatment for the paraquat assay. (b,c) Treatment with

UA from the eggs stage extends lifespan of worms exposed to paraquat 4 mM from Day 1

(D1) (b) and 8 (D8) (c). The experiment was stopped at day 14 of adulthood to avoid any

interference with the mortality due to aging. (d) UA has no effect on ROS production as

revealed by the Mitosox® staining (n = 6). Values are mean ± s.e.m. ns: not significant. (e)

UA extends lifespan of worms treated during adulthood in presence of N-acetylcysteine

(NAC) at 5 mM. Data are representative of at least two independent experiments. UA was

used at 50 µM. Vehicle corresponds to 1% DMSO. See also Supplementary Table 1.



Supplementary Fig. 6a

Fold

incr

ease

(p-A

MP

Kα/

AM

PK

α)

vehicle 10 20 50UA (μM)



**

*** **

b



** *****


***

0

5

10

15

20

0

1

2

3

4

5

0

20

40

60

0

5

10

15

0

1

2

3

4

5

05

10152025

100300500

0

1

2

3

4

5


**

0

1

2

3

4

5


**

***

c

vehicleUA 10 μM

vehicleUA 50 μM

vehicleUA 100 μM

Cou

ntC

ount

Cou

nt

FITC-A

FITC-A

FITC-A

samplevehicle

UA 10 μM

mean8.95

9.09

samplevehicle

UA 50 μM

mean8.95

14.5

samplevehicle

UA 100 μM

mean8.95

17.8

d

e

g

0

2

4

6

8

0

2

4

6

8

0

2

4

6

8

0

5

10

15

0

0.5

1.0

1.50 6 12 24

0 6 12 24

0 6 12 24

0 6 12 24

0 6 12 24

Fold

incr

ease

(SD

HB

(CII)

/ TO

M20

)

*

*

*

*

*

0.8

0.9

1.0

1.1

1.2

Oxy

gen

cons

umpt

ion

(rat

io o

ver c

ontro

l)

vehicleUA 0.1 µM

UA 1 µMUA 10 µM

10-8 10-7 10-6 10-5 10-4 10-30

0.5

1.0

1.5

UA concentration (M)

f

0 20 40 60 80 100 120Time (min)

Injection of the compound

Oxy

gen

cons

umpt

ion

(rat

io o

ver c

ontro

l)

Fold

incr

ease

(LC

3-II/

LC3-

I)

Fold

incr

ease

(p62

/TO

M40

)

Fold

incr

ease

(Ub/

TOM

40)

Fold

incr

ease

(p-A

MP

Kα/

AM

PK

α)

Fold

incr

ease

(LC

3-II/

LC3-

I)

Fold

incr

ease

(p62

/TO

M40

)

Fold

incr

ease

(Ub/

TOM

40)

Fold

incr

ease

(UQ

CR

C2

(CIII

) / T

OM

20)

Fold

incr

ease

(MTC

O1

(CIV

) / T

OM

20)

Fold

incr

ease

(ATP

5A (C

V) /

TO

M20

)Fo

ld in

crea

se(T

OM

20 /

HS

P90

)

Fluorescence intensity

4Time (h)

12 241Geo

met

ric M

ean

(103 )

3

2

1

0

4

*** *****

vehicle

UA

Time (h)

Supplementary Figure 6 The effect of UA on the mitophagy is conserved in mammalian

cells. (a,b) Pixel intensity quantification of the WB shown in the Fig. 4a (a) and Fig. 4b (b).



(c) FACS analyses demonstrated a dose dependent induction of UA-induced autophagy, as

measured by LC3-II accumulation, in C2C12 myoblasts after 24 hours treatment (n = 3). (d)

Quantification of the fluorescence intensity of the CytoID® dye by FACS shown in the Fig. 4d

(n = 4). (e) UA does not affect respiration of the C2C12 myotubes in the two hours following

the addition of the compound at concentrations ranging from 0.1 to 10 µM (n = 5). Grey area

represents the 95% confidence interval of the vehicle group. (f) UA does not impact

respiration of mitochondria isolated from mouse liver, even when used at doses up to 100 µM.

Grey area represents the 95% confidence interval of the vehicle group. (g) Pixel intensity

quantification of the WB shown in the Fig. 5i (n = 3). CII: complex II; CIII: complex III; CIV:

complex IV; CV: complex V. Values are mean ± s.e.m. Data are representative of at least

two independent experiments. *P ≤0.05; **P ≤0.01; ***P ≤0.001 by one-way ANOVA. Vehicle

corresponds to 0.1% DMSO.



Supplementary Fig. 7[U

A] (n

g / m

l)

0 2 7

Treatment duration(days)

n.d.

a b

0.85

0.90

0.95

1.00

0

0.05

0.10

0.15

0.20

Treatment duration (weeks)

0 6Lean

mas

s / b

ody

wei

ght

Fat m

ass

/ bod

y w

eigh

t


0 6

e

0

2

4

6

8

10

Velo

city

(cm

/s)

fControlUA

ControlUA

ControlUA

h

Control UA

CI

CIV

CIV

CII

Control UA

Control UA Control UA

Control UA Control UA

IVIII

IV

II

i

j

0 10 20 3030

35

40

45

50

55

Body

wei

ght (

g)

0 10 20 3010

20

30

40

Fat m

ass

(%)

0 10 20 30

60

70

80

90

Lean

mas

s (%

)

0

1

2

3

4

0

5

10

15

20

0 2 7

Treatment duration(days)

[UA]

(ng

/ g)

n.d.




cControlUA

ControlUA

ControlUA

-2 0 2 4 6 826

28

30

32

34

Body

wei

ght (

g)


ControlUA

d

0.5 1 2 4Ratio over control

g

Control

UAMyh6

Myh2

Myh4

Myh1

Supplementary Figure 7 Orally delivered UA is bioavailable in muscle and does not impact

muscle mass. (a,b) The native form of UA is present in plasma (a) and muscle (b) of rats

after 2 and 7 days of compound administration by food admix (n = 6). n.d.: not detectable. (c)



UA has no effect on body weight gain, fat mass gain and lean mass loss in 16-months old

male C57BL6/J mice treated for 34 weeks with UA admixed to high-fat diet (HFD) (n = 25).

(d,e) UA did not affect body weight (d), lean mass or fat mass (e) in 24-months old male

C57BL6/J mice treated during 6 weeks with UA admixed to normal chow diet (NCD) (n = 16).

(f) Young rats were randomized according to their velocity measured during an Elevated Plus

Maze test at the age of 5.5 weeks, before starting the treatment (n = 12). (g) UA does not

change the expression of transcripts of myosin heavy chain isoforms in gastrocnemius

muscle of 24-months old male C57BL6/J mice treated for 34 weeks under HFD (n = 10). (h)

Images of BN-PAGE for the quantification of respiratory complexes proteins abundance

shown in Fig. 6h. (i,j) Images of the complexes activity measured in BN-PAGE gel for

complex II (i) and complexes I and IV (j) shown in Fig. 6h. Values are mean ± s.e.m. Control

means the corresponding diet without UA admixed.



Supplementary Figure 8 Summary of the effects of UA at the organelle and molecular

levels. (a) Overview of the effects of UA observed in vitro. UA triggers mitochondrial fission

and decreases mitochondrial membrane potential (∆ψm). This leads to stabilization of Pink1

and ubiquitination of mitochondrial proteins. The ubiquitin signal is recognized by the

autophagosome adaptor p62, which is recruited at the mitochondria. In parallel, an unknown

upstream signal triggers autophagy and the conversion of LC3-I into LC3-II. This leads to the

formation of autophagosomes, and autolysosomes after the fusion with lysosomes. (b−d)

Overview of the effects of UA on mitochondrial metabolism. Following the induction of

mitophagy by UA, mitochondria shift their metabolism toward complex II-driven respiration

(b). This is evidenced by the fact that in glycolytic conditions, i.e. when glucose or substrates

of aerobic glycolysis are in excess, UA decreases oxygen consumption (c). Conversely,

when cells are grown in oxidative conditions, i.e. when fatty acids are in excess, UA

increases oxygen consumption (d).


summary of the c. elegans lifespan experiments all rnai feeding conditions and different treatments...

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