RESEARCH ARTICLE
Gene ic and cellula sensi i i y o Caeno habdi is elegans o he
chemo he apeu ic agen cispla in
F ancisco Ja ie Ga cı
a-Rod ı
guez
1,2
, Ca men Ma ı
nez-Fe nandez
1
, Da id B ena
1
, Dmy o Kukh a
1
,
Xenia Se a
1
, E nes Nadal
3
, Mike Boxem
4
, Sebas ian Honnen
5
, An onio Mi anda-Vizue e
6
,
Albe o Villanue a
2,
*and Julian Ce on
1,
*
ABSTRACT
Cispla in and de i a i es a e commonly used as chemo he apeu ic
agen s. Al hough he cy o oxic ac ion o cispla in on cance cells is
e y e icien , clinical oncologis s need o deal wi h wo majo
di icul ies, namely he onse o esis ance o he d ug and he
cy o oxic e ec in pa ien s. He e, we used Caeno habdi is elegans o
in es iga e ac o s in luencing he esponse o cispla in in mul icellula
o ganisms. In his he maph odi ic model o ganism, we obse ed ha
spe m ailu e is a majo cause o cispla in-induced in e ili y. RNA
sequencing da a indica e ha cispla in igge s a sys emic s ess
esponse, in which DAF-16/FOXO and SKN-1/NRF2, wo conse ed
ansc ip ion ac o s, a e key egula o s. We de e mined ha inhibi ion
o he DNA damage-induced apop o ic pa hway does no con e
cispla in p o ec ion o he animal. Howe e , mu an s o he p o-
apop o ic BH3-only gene ced-13 a e sensi i e o cispla in, sugges ing
a p o ec i e ole o he in insic apop o ic pa hway. Finally, we
demons a ed ha ou sys em can also be used o iden i y mu a ions
p o iding esis ance o cispla in and he e o e po en ial bioma ke s o
inna e cispla in- e ac o y pa ien s. We show ha mu an s o he
edox egula o x -1, o holog o he mammalian hio edoxin
educ ase 1 TRXR1, display cispla in esis ance. By CRISPR/Cas9,
we de e mined ha such esis ance elies on he p esence o he
single selenocys eine esidue in TRXR-1.
This a icle has an associa ed Fi s Pe son in e iew wi h he i s
au ho o he pape .
KEY WORDS: Caeno habdi is elegans, RNA-seq, Cispla in
INTRODUCTION
The US Food and D ug Adminis a ion app o ed he use o cispla in
[CDDP, cis-diammine-dichlo opla inum(II)] as a chemo he apeu ic
agen in 1978. Since hen, cispla in and o he pla inum-based
de i a i es ha e been used success ully in cance ea men . To
illus a e hei impac in he clinic, i has been es ima ed ha
app oxima ely hal o all pa ien s unde going chemo he apeu ic
ea men ecei e a pla inum d ug (Galanski, 2006). Cispla in exe s
ac i i y agains a wide spec um o solid neoplasms, including
es icula , bladde , o a ian, head and neck, gas ic and lung cance s
(Dasa i and Be na d Tchounwou, 2014). S ikingly, es icula
cance was p e iously a al, bu ea men wi h cispla in p o ided a
cu e o 80% o he pa ien s (Gonzalez-Exposi o e al., 2016).
Despi e i s e ec i eness, he e a e pa ien s in insically esis an o
cispla in-based he apies, and an impo an ac ion o umo s
e en ually de elop chemo esis ance (Amable, 2016).
Cispla in is composed o a double-cha ged pla inum ion
su ounded by ou ligands, wo amines and wo chlo ides. Inside
cells, he low chlo ide concen a ion acili a es cispla in aqua ion,
eplacing chlo ide g oups by wa e molecules. This p ocess p oduces
a hyd olyzed (o aqua ed) o m o cispla in ha is a po en elec ophile
(a ac ed o elec ons) ha can eac wi h any nucleophile, including
nucleic acids and he sul hyd yl g oups o p o eins (Kelland, 2007).
Cispla in ac i i y has an impac in he nucleus and in he
cy oplasm. In he nucleus, cispla in p oduces DNA in a- and
in e s and c osslinks ha lead o apop osis (Kelland, 2007). In he
cy oplasm, owing o i s elec ophilic ac i i y, cispla in beha es as an
oxidan (loss o elec ons esul s in oxida ion), binding o p o eins,
including mi ochond ial p o eins, and especially o hiol g oups
(-SH). Thus, cispla in p oduces a eac i e oxygen species
homeos asis imbalance ha leads o mo e oxidizing condi ions,
which iola e no mal cellula unc ion and, ul ima ely, can also
p omo e apop osis (Wang and Lippa d, 2005). As a esul o i s
b oad and unspeci ic mode o ac ion, cispla in also a ec s no mal
cells. Neph o oxici y, neu o oxici y and o o oxici y a e some o he
dose-limi ing side e ec s epo ed upon cispla in he apy (Kelland,
2007). Ne e heless, he majo obs acle o he clinical e icacy o
cispla in as an an icance d ug is he chemo esis ance de eloped by
umo s a he han i s oxici y in no mal cells.
The acquisi ion o cispla in esis ance is mul i ac o ial. The
mechanisms by which umo cells become esis an o he ac ion o
cispla in ha e been classi ied in o h ee ypes (Galluzzi e al., 2014):
(1) p e- a ge mechanisms, i.e. educing in acellula accumula ion
o cispla in o inc easing seques a ion o cispla in by nucleophilic
sca enge s as glu a hione (GSH), me allo hioneins and o he
cys eine- ich p o eins; (2) on- a ge mechanisms, i.e. acqui ing he
abili y o epai adduc s o becoming ole an o un epai ed DNA
lesions; and (3) pos - a ge mechanisms, i.e. hampe ing he
execu ion o apop osis in esponse o DNA damage (Siddik, 2003).
Recei ed 8 Janua y 2018; Accep ed 10 Ap il 2018
1
Modeling human diseases in C. elegans. Genes, Diseases and The apies
P og am, Bell i ge Biomedical Resea ch Ins i u e - IDIBELL, L’Hospi ale de
Llob ega , 08908 Ba celona, Spain.
2
P og am Agains Cance The apeu ic
Resis ance (P oCURE), Ca alan Ins i u e o Oncology (ICO), Bell i ge Ins i u e o
Biomedical Resea ch (IDIBELL), L’Hospi ale del Llob ega , 08908 Ba celona,
Spain.
3
Tho acic Oncology Uni , Depa men o Medical Oncology, Ca alan Ins i u e
o Oncology (ICO), L’Hospi ale de Llob ega , 08908 Ba celona, Spain.
4
Di ision o
De elopmen al Biology, Depa men o Biology, Facul y o Science, U ech
Uni e si y, 3584 CH U ech , The Ne he lands.
5
Hein ich Heine Uni e si y
Du
sseldo , Medical Facul y, Ins i u e o Toxicology, D-40225 Du
sseldo , Ge many.
6
Ins i u o de Biomedicina de Se illa, Hospi al Uni e si a io Vi gen del Rocı

o/CSIC/
Uni e sidad de Se illa, E-41013 Se illa, Spain.
*Au ho s o co espondence ([email p o ec ed]; [email p o ec ed])
F.J.G., 0000-0001-9933-1990; C.M., 0000-0003-1544-4387; D.B., 0000-0003-
3318-5120; A.V., 0000-0002-4164-6671; J.C., 0000-0003-4739-2243
This is an Open Access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion
License (h p://c ea i ecommons.o g/licenses/by/3.0), which pe mi s un es ic ed use,
dis ibu ion and ep oduc ion in any medium p o ided ha he o iginal wo k is p ope ly a ibu ed.
1
© 2018. Published by The Company o Biologis s L d
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Disease Models & Mechanisms (2018) 11, dmm033506. doi:10.1242/dmm.033506
Disease Models & Mechanisms
Caeno habdi is elegans is a well-es ablished o ganism o he
s udy o signaling pa hways in esponse o d ug exposu e (Kale a
and Henga ne , 2006). P e ious s udies, pe o med in dis inc
biological con ex s (Hemmingsson e al., 2010; Collis e al., 2006),
ha e e ealed he alue o C. elegans o iden i y genes ela ed o he
cispla in esponse (Table S1). He e, we show, o he i s ime, ha
cispla in in C. elegans p oduces DNA adduc s and a sys emic
esponse d i en by wo conse ed ansc ip ion ac o s. We ha e
unco e ed a spe m-speci ic sensi i i y o cispla in and ha e
demons a ed ha esis ance o he nema ode o cispla in elies on
he p esence o a single selenocys ein o he hio edoxin educ ase
TRXR-1. Thus, his epo is a mo e comp ehensi e s udy o he
global esponse o C. elegans o cispla in ha also es ablishes a
eliable me hodology o u u e s udies on mechanisms o esis ance
o cispla in ha would con inue con ibu ing o he sea ch o new
a ge s and ma ke s o he bene i o cispla in-based he apies.
RESULTS
A eliable assay o s udy he e ec o cispla in in C. elegans
In C. elegans nema odes, cispla in p oduces a wide a ie y o
pheno ypes depending on he concen a ion, leng h o ea men and
de elopmen al s age o he ea ed animals (Tables S1 and S2).
To implemen a eliable me hodology and sys ema ically in es iga e
he esponse o C. elegans o cispla in, we es ablished dose–
esponse pa e ns du ing la al de elopmen , when cell di isions
occu in soma ic and ge m cells. We exposed a synch onized
popula ion o L1 la ae on nema ode g ow h medium (NGM) pla es
o di e en cispla in concen a ions o 96 h. We obse ed e ec s
anging om a de elopmen al delay a 50 µg/ml o a la al a es a
200 µg/ml (Fig. 1A). Based on his assay, we concluded ha body
leng h a 48 h pos -L1, upon cispla in exposu e om 50 o 75 µg/ml,
is a eliable indica o o he e ec o cispla in du ing C. elegans
de elopmen (Fig. 1B). Thus, we es ablished a me hodology o
in es iga e how dis inc ea men s o gene ac i i ies can in luence
he esponse o C. elegans o cispla in.
T ansc ip ional esponse o C. elegans o cispla in
To explo e he global esponse o nema odes o cispla in, we s udied
ansc ip ional signa u es in RNA sequencing (RNA-seq)
expe imen s. We pe o med his new ansc ip ome analysis using
a mixed-s age nema ode popula ion ha was ea ed o 24 h wi h
60 µg/ml o cispla in, because his dosage o cispla in p oduces
some pheno ypes, bu does no comp omise he iabili y o he
animals. To educe he numbe o alse posi i es, we used wo
Fig. 1. Dose– esponse e ec o
cispla in on C. elegans de elopmen
and RNA-seq o animals exposed
o cispla in. (A) E ec o dis inc
concen a ions o cispla in on la al
de elopmen . Synch onized L1 la ae
we e g own on aga pla es and exposed o
di e en doses o cispla in o 96 h a
20°C. Body leng h alues ep esen mean
and SD (n=50) o wo di e en
expe imen s. (B) Body leng h o
nema odes ha we e g own om L1
s age exposed o di e en doses o
cispla in o 48 h a 20°C. Ba s ep esen
mean and SD o h ee independen
eplica es (n=50, N=3). (C) Schema ic
ep esen a ion o ansc ip omic analyses.
Wild- ype C. elegans om mixed s ages
we e cul u ed wi h and wi hou cispla in
(60 µg/ml) o 24 h a 20°C. Then, o al
mRNA o wo biological eplica es was
pu i ied and sequenced. Volcano plo
ep esen s genes signi ican ly up- and
down egula ed (P<0.01) in cispla in-
ea ed e sus un ea ed animals.
(D) Colo code ep esen s he unc ional
ca ego ies o up- o down egula ed genes.
Genes we e clus e ed acco ding o he
gene unc ional classi ica ion ool DAVID
and GO e ms e ie ed om Wo mBase.
2
RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033506. doi:10.1242/dmm.033506
Disease Models & Mechanisms
biological eplica es o compa e he ansc ip omes o ea ed and
non- ea ed popula ions (Fig. 1C). We used he Cu links algo i hm
(T apnell e al., 2012) o p ocess he RNA sequencing da a and
s udy di e en ial exp ession o genes. We iden i ied a se o 83
genes up egula ed and 78 genes down egula ed by cispla in in bo h
expe imen s (P≤0.05; Table S3). A mo e es ic ed lis o
candida es, using a cu -o P- alue o ≤0.01, included 28 genes
up egula ed and 29 down egula ed upon cispla in exposu e.
To explo e he unc ions o hese 57 genes, we pe o med a gene
on ology (GO) analysis and iden i ied p edic ed p o ein domains
(COG) (Ta uso e al., 2003; Huang e al., 2009) (Fig. 1D). Genes
encoding CUB-like domains, C- ype lec ins and glu a hione-S-
ans e ases we e ound among he genes up egula ed upon cispla in
exposu e. In C. elegans, he exp ession o such genes is associa ed
wi h de oxi ica ion, edox balance, s ess esponse and inna e
immune sys em, and i is o en egula ed by he ansc ip ion ac o s
DAF-16 and SKN-1 (Singh and Aballay, 2009; Pa k e al., 2009;
Yen e al., 2011; Teppe e al., 2013) (Table S4). We also de ec ed an
o e lap wi h genes induced upon ionizing adia ion (IR) (G eiss
e al., 2008; Schumache e al., 2005a,b) (Table S4). Among hose,
we ound genes in ol ed in he apop o ic signaling cascade,
including egl-1 and ced-13 (G eiss e al., 2008). Howe e , as
desc ibed below, hese wo genes unc ion in a dis inc manne upon
cispla in exposu e.
O he 29 genes down egula ed by cispla in, we ound ha mos
o hose we e spe m-speci ic genes (O iz e al., 2014), wi h he
Majo Spe m P o ein (MSP) domain as he mos equen GO
anno a ion (Table S5). The e o e, he educed b ood size induced by
cispla in migh be p o oked, a leas in pa , by a speci ic e ec o
his agen on spe ma ogenesis and/o on spe m ac i i y.
Cispla in educes he ge m cell pool and a ec s spe m
unc ionali y
Ou ansc ip ome analyses indica ed ha male ge mline genes a e
pa icula ly down egula ed in he p esence o cispla in. To
in es iga e he e ec o cispla in on he ge mline, we exposed L4
animals o cispla in o 24 h, because he swi ch om
spe ma ogenesis o oogenesis occu s a his s age and he e o e
bo h p ocesses can be a ec ed (Ellis and Schedl, 2007). As
expec ed, we obse ed a dose-dependen educ ion o he b ood
size upon cispla in exposu e (Meie e al., 2014) (Fig. 2A). This
educ ion was co ela ed wi h an inc ease in he numbe o
un e ilized eggs laid (Fig. 2A) and a dec ease in he numbe o
cells a he p oli e a i e egion o he ge mline (Fig. 2B). We
obse ed ha cispla in- ea ed ge mlines displayed ewe nuclei in
he mi o ic egion ha appea ed o be bigge (Fig. 2C), which is an
e ec desc ibed in ge mlines exposed o DNA-damaging agen s
ha is a ibu able o cell cycle a es upon ac i a ion o he S-phase
checkpoin (Ga ne e al., 2004).
The inc eased numbe o un e ilized oocy es is a pheno ypic
hallma k o spe m ailu e in C. elegans (Kadandale and Singson,
2004). To in es iga e whe he he un e ilized oocy es obse ed
upon cispla in ea men we e he esul o de ec i e spe m, we
c ossed cispla in- ea ed he maph odi es wi h un ea ed males. We
obse ed ha spe m om un ea ed males escued, a leas in pa ,
he b ood size and ab oga ed he p esence o un e ilized oocy es
(Fig. 2D). These esul s sugges ha he excess o un e ilized
oocy es is a ibu able o he e ec o cispla in in spe ma ogenesis
a he han in oogenesis.
To in es iga e he e ec o cispla in on spe m unc ionali y, we
used he emale/male s ain og-2(oz40), allowing he ea men o
males and emales independen ly be o e he c oss. og-2(oz40)
emales exposed o cispla in and c ossed wi h og-2(oz40) un ea ed
males showed a educed b ood size, as expec ed om he e ec o
cispla in on mi o ic cells, bu we did no de ec an inc eased
p oduc ion o un e ilized oocy es (Fig. 2E). On he con a y, by
c ossing og-2(oz40) males exposed o cispla in wi h un ea ed
og-2(oz40) emales we obse ed a s onge educ ion o he
p ogeny, bu also a highe numbe o un e ilized oocy es. These
esul s con i m ha , a a simila dose, cispla in does no hampe he
capaci y o oocy es o be e ilized bu a ec s he capabili y o spe m
o e ilize.
In summa y, he exposu e o cispla in p oduces smalle ge mlines
as consequence o a educ ion o he p oli e a i e egion and
e iliza ion p oblems caused by he e ec o cispla in in he spe m
(Fig. 2F).
DAF-16 and SKN-1 a e key playe s in he esponse o
cispla in
The abundance o dod (downs eam-o -da -16) genes and o he
s ess esponse genes in he lis o genes up egula ed upon cispla in
exposu e led us o s udy he ole o DAF-16 and SKN-1. These a e
he o hologs o human FOXO3 and NRF2, espec i ely, and a e
equi ed o s ess esis ance in C. elegans (Rod iguez e al., 2013).
S ikingly, we ound ha hal o he up egula ed genes we e
p e iously epo ed as DAF-16 and/o SKN-1 a ge s (Fig. 3A).
The e olu iona ily conse ed insulin/insulin-like g ow h ac o
signaling (IIS) pa hway, h ough i s main ansc ip ion ac o DAF-
16/FOXO, con ols many di e en biological p ocesses and
egula es a wide a ie y o s esses, including s a a ion, oxida i e
s ess (Honda and Honda, 1999), hea y me al oxici y (Ba sy e
e al., 2001) and ul a iole adia ion (Mu akami and Johnson,
1996). DAF-16 is cons i u i ely exp essed and seques e ed in
he cy oplasm in i s phospho yla ed o m. I he inhibi o y
phospho yla ion is comp omised, e.g. by educed signaling o he
DAF-2 ecep o y osine kinase o s ess condi ions, DAF-16 will
ansloca e o he nucleus. Using a DAF-16::GFP epo e s ain, we
obse ed ha DAF-16 nuclea loca ion inc eased upon cispla in
exposu e in a dose-dependen manne (Fig. 3B), con i ming he
implica ion o he IIS pa hway in he esponse o cispla in.
Nex , we in es iga ed he ex en o which he manipula ion o he
IIS ac i i y could modi y he esponse o he o ganism o cispla in.
We obse ed ha da -2(e1370) mu an s, which a e animals lacking
a unc ional DAF-2/IGF-1-like ecep o ha cons i u i ely induce
DAF-16 nuclea localiza ion (Yen e al., 2011), display esis ance o
cispla in du ing la al de elopmen (Fig. 3C). Consis en ly, da -
16(mu86) mu an s we e hype sensi i e o cispla in (Fig. 3C),
highligh ing he ele ance o DAF-16 nuclea ac i i y in he cellula
esponse o cispla in.
Di e en ly om da -2 and da -16,skn-1 is an essen ial gene
hampe ing he use o loss-o - unc ion mu an s. To con i m he
implica ion o SKN-1 in he esponse o cispla in, we used a GFP
epo e o one o i s canonical a ge s, gs -4. SKN-1 egula es he
s ess-induced gs -4 ansc ip ion in he p esence o edox ac i e
compounds, such as pa aqua o hea y me als (Tawe e al., 1998;
Roh e al., 2006). In ou RNA-seq da a, gs -4 is one o he SKN-1-
egula ed genes s ongly induced by cispla in, and we alida ed his
esul using a gs -4 ansc ip ional GFP epo e (Fig. 3D,E).
Mo eo e , using skn-1 RNA in e e ence (RNAi) we con i med ha
he cispla in induc ion o gs -4 exp ession was skn-1 dependen and
da -16 independen (Fig. 3D,E). Thus, by s udying he impac o
cispla in in he global gene exp ession we unco e ed a sys emic
esponse o he o ganism ha is d i en by wo conse ed
ansc ip ion ac o s. The ac ha hese ansc ip ion ac o s a e
3
RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033506. doi:10.1242/dmm.033506
Disease Models & Mechanisms
e ec o s o me abolic and en i onmen al signals opens new
a enues o egula e he esponse o cispla in in mul icellula
o ganisms.
The BH3-only p o ein CED-13 p o ec s agains cispla in
We demons a e, o he i s ime, ha cispla in leads o he
o ma ion o DNA adduc s in C. elegans (Fig. 4A) and induces
he exp ession o wo apop osis- ela ed genes, egl-1 and ced-13,
which encode BH3-only p o eins, which a e a subse o he Bcl-2
amiliy ha con ain only a BH3 domain and p omo e apop osis.
egl-1 and ced-13 a e ansc ip ionally induced upon DNA damage
(also upon exposu e o UV o IR; S e giou e al., 2007), and his
induc ion is CEP-1/P53 dependen (G eiss e al., 2008). Y47G7B.2,
ano he cispla in-induced gene in ou RNA-seq, encodes a
nema ode-speci ic gene ha is also up egula ed upon DNA
damage in a cep-1-dependen manne (G eiss e al., 2008). Thus,
he e ec s o cispla in on up egula ion o apop o ic genes could be
a ibu able o cep-1-dependen DNA damage.
egl-1 is equi ed o DNA damage-induced apop osis in bo h
soma ic issues and he ge mline (Ga ne e al., 2000). We
in es iga ed egl-1 induc ion upon cispla in exposu e in he
soma using a GFP epo e s ain (egl-1p::GFP). Du ing la al
de elopmen , only 18 soma ic cells o wild- ype nema odes
unde go apop osis a he ea ly L2 s age (Le e and Henga ne ,
2006). S ikingly, a 24 h cispla in ea men in C. elegans ( om L1
o L2) induced ec opic egl-1p::GFP exp ession ha was e iden in
soma ic cells (Fig. 4B,C). Mo eo e , his cispla in-induced egl-1
ec opic exp ession was, a leas in pa , cep-1 dependen (Fig. 4D).
In he canonical DNA damage-induced apop o ic pa hway, cep-1
is ups eam o he BH3-only p o eins EGL-1 and CED-13.
Howe e , ced-13, and i s downs eam gene ced-3,ha ean
addi ional ole in he in insic apop o ic pa hway ha p omo es
Fig. 2. E ec o cispla in in he ge mline. (A) Cispla in educes b ood size and inc eases he pe cen age o un e ilized oocy es laid. L4 animals we e exposed o
cispla in o 24 h a he indica ed concen a ions (n=12, N=2). (B) Cispla in educes he numbe o ge m cells in he p oli e a i e egion o C. elegans ge mline.
Numbe o cells co esponds o nuclei obse ed, in a single z-s ack, a he p oli e a i e egion (50 μm away om he dis al end o he gonad) (n=15, N=2).
(C) Rep esen a i e DAPI s aining o young adul ge mline exposed o cispla in o 24 h. Righ panels show magni ica ion o he ge mline p oli e a i e egion
(highligh ed a ea). (D) Impac o cispla in on e iliza ion. Cispla in p oduces un e ilized oocy es and causes educed b ood size in sel - e ilized he maph odi e
wo ms (‘Sel ’), bu hise ec is escued, in pa , by c ossing cispla in- ea ed he maph odi es wi h un ea ed males (‘C oss’;n=12, N=2). ***P>0.001, **P>0.01, *P>0.1;
n.s., non-signi ican . (E) Cispla in-induced un e ilized oocy es a e caused by de ec i e spe m. og-2(oz40) he maph odi es do no p oduce spe m, and ma ing is
necessa y o main ain he s ain. Cispla in p oduces un e ilized oocy es only when males a e ea ed (n=12, N=2).
++
P>0.01,
+
P>0.1 compa ed wi h emale ea men
in he same condi ions. (F) Schema ic diag am o illus a e he e ec o cispla in in he ge mline: bigge nuclei a he p oli e a i e zone (ligh blue), de ec i e spe m
( ed do s) and un e ilized oocy es (blue a ow). Ba s show mean and s.e.m., and S uden ’s - es was applied.
4
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p o ec i e changes a he han killing damaged i epa able cells
(Yee e al., 2014). Acco dingly, we ound ha ced-13 and ced-3
mu an s we e sensi i e o cispla in, whe eas wo di e en cep-1
mu an s we e no esis an o cispla in (Fig. 4E,F).
In summa y, du ing C. elegans la al de elopmen , cispla in
induces he exp ession o egl-1 and ced-13, bu hei in luence is
dis inc . Inhibi ion o he canonical DNA damage-induced apop o ic
pa hway by using cep-1 mu an s does no p oduce animals esis an
o cispla in. On he con a y, ced-13 and ced-3 mu an s a e sensi i e
o cispla in, unde sco ing a p o ec i e ole o he in insic apop o ic
pa hway upon cispla in exposu e in soma ic cells.
TRXR-1 ac i i y con e s sys emic sensi i i y o cispla in
The majo i y o C. elegans genes ela ed o cispla in esponse
published o da e p esen cispla in sensi i i y i inac i a ed by RNAi
o mu a ion (Table S1). The human o hologs o hese genes a e
po en ial a ge s o he apies o e-sensi ize cispla in- esis an
umo s. Gi en he clinical impo ance o iden i ying e ac o y
pa ien s, we wonde ed whe he ou sys em was also aluable o he
s udy o genes whose inac i a ion con e s esis ance o cispla in. In
he clinic, mu a ions o single nucleo ide polymo phisms in hese
genes could unc ion as p edic i e ma ke s o cispla in esponse. In
human cell lines, hio edoxin educ ase 1 (TRXR1, also known as
TXNRD1) selenocys eine amino acid is a di ec a ge o cispla in,
p oducing cy o oxic and highly p o-oxidan TRXR1 o ms called
SecTRAPs, which lead o high edox s ess esul ing in cell dea h
(Anes ål e al., 2008). TRXR1 is one o he ew selenop o eins
[p o eins ha include a selenocys eine (SeCys) amino acid] in
mammals, bu TRXR-1 is he only selenop o ein in C. elegans (Li
e al., 2012) and, in con as o mammals, his p o ein is no essen ial
Fig. 3. Role o DAF-16/FOXO and SKN-
1/N 2 ansc ip ion ac o s in he
esponse o cispla in. (A) Venn
diag ams showing he signi ican o e lap
be ween he genes induced by cispla in
ound in ou ansc ip omic analyses and
genes egula ed by DAF-16 (Teppe e al.,
2013) and SKN-1 (Oli ei a e al., 2009).
P<0.001 by Fishe ’s es . (B) Cispla in
induces DAF-16 nuclea ansloca ion.
Rep esen a i e image o L2 la ae
ca ying a ansla ional GFP epo e o
DAF-16 wi h and wi hou cispla in
(90 µg/ml o 5 h). The g aph ep esen s
he pe cen age o synch onized L2 la ae
wi h DAF-16::GFP p edominan ly nuclea
a e 5 h o exposu e o cispla in a he
indica ed concen a ions. Ba s ep esen
mean and s.e.m. (n=50, N=3). **P<0.01
ela i e o un ea ed wo ms by S uden ’s
- es . (C) IIS pa hway in luences he
esponse o cispla in. Body leng h o
synch onized L1 la ae g own o 4 days in
he absence o p esence o cispla in.
Wo ms we e g own a 15°C o a oid da -
2(e1370) daue pheno ype. The
inac i a ion o he IIS pa hway in da -
2(e1370) mu an s, which keeps DAF-16
cons i u i ely in he nucleus, causes
esis ance o cispla in, whe eas he da -
16(mu86) null mu an inc eases cispla in
sensi i i y. T anslesion syn hesis
polyme ase 1 mu an allele polh-1(i 31)
was used as a posi i e con ol (Roe ink
e al., 2012). Ba s ep esen mean and SD
(n=50). ***P<0.001 ela i e o un ea ed
wo ms by S uden ’s - es . (D) Cispla in-
induced ac i a ion o gs -4 is egula ed by
SKN-1. Rep esen a i e images o
synch onized gs -4p::GFP L4/young adul
animals g own a 20°C on da -16(RNAi)
o skn-1(RNAi) bac e ia om he L1 s age
in he p esence o absence o cispla in.
skn-1(RNAi) inhibi s he cispla in-induced
gs -4 exp ession. Wo ms ed wi h g p
RNAi we e used as a posi i e con ol.
(E) Fluo escence in ensi y o each
condi ion. Ba s ep esen he mean. E o
ba s indica e he s.e.m. (n=30, N=2).
***P<0.001 ela i e o un ea ed animals
by S uden ’s - es .
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o iabili y (S en all e al., 2011). Deple ion o C. elegans x -1
does no p oduce pheno ypes in e ms o mo phology, g ow h,
li espan, b ood size o esponse o oxida i e s ess. I is essen ial
only o la al mol ing when he glu a hione educ ase gene gs -1 is
inac i a ed in pa allel (S en all e al., 2011). All hese ea u es
make C. elegans an excellen mul icellula sys em in which o
s udy speci ically he ole o he SeCys esidue in he esponse
o cispla in.
Fig. 4. Role o apop o ic pa hways in he esponse o C. elegans o cispla in. (A) Sou he n blo showing ha 1,2-GpG-in as and c osslinks a e aised upon
cispla in exposu e o a C. elegans popula ion. The e is a dose-dependen accumula ion o DNA adduc s in he p esence o cispla in. NRK-52E a cells, in which
1,2-GpG-in as and c osslinks a e no p esen (K u
ge e al., 2016), we e incuba ed wi h cispla in as a posi i e con ol. (B) Cispla in induces egl-1 ec opic
exp ession in soma ic issues du ing la al de elopmen . Rep esen a i e images o L2 wo ms ca ying a egl-1p::GFP epo e , un ea ed and ea ed wi h cispla in
(60 µg/ml o 24 h a 20°C). (C) Quan i ica ion o wo ms wi h ec opic egl-1p::GFP luo escence in soma ic cells o synch onized L1 la ae g own a he indica ed
concen a ions o cispla in o 24 h a 20°C. Ba s ep esen mean and s.e.m. o h ee di e en expe imen s (n=50). **P<0.01 by S uden ’s - es . (D) egl-1 ec opic
induc ion in soma ic cells equi es cep-1/p53. Quan i ica ion o cells wi h ec opic egl-1p::GFP luo escence o L1 la ae exposed o 60 µg/ml o cispla in o
24 h a 20°C in wild- ype and cep-1(gk138) mu an s. Ba s ep esen mean and s.e.m. o h ee di e en expe imen s (n=50). ***P<0.01 by S uden ’s - es .
(E,F) Inac i a ion o he cep-1/p53 apop o ic pa hway does no inc ease cispla in esis ance. (E) Body leng h o L1 la ae o wild- ype and dis inc mu an alleles
g own in cispla in (60 µg/ml) o 48 h a 20°C. Ba s ep esen mean and s.d. (n=50). This expe imen was pe o med h ee imes wi h simila esul s. (F) Rela i e
alues o g ow h delay we e ob ained by calcula ing he pe cen age o he di e ence in he body leng h be o e and a e cispla in exposu e (non-pa ame ic
K uskal–Wallis es , ***P<0.001; *P< 0.05; n.s., non-signi ican ).
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We es ed he obus ness o ou model by s udying he ole o
hio edoxin educ ase 1 ( x -1)in heC. elegans esponse o cispla in.
We used he CRISPR/Cas9 sys em o p oduce wo endogenous
TRXR-1 a ian s, x -1(ce 4[U666C])and x -1(ce 5[U666STOP]),
ha change he selenocys eine o cys eine and a STOP codon,
espec i ely (Fig. 5A). In a simila manne o he null mu an x -
1(s 47), hese missense mu a ions showed la al a es , a ibu able o
mol ing de ec s, in combina ion wi h gs -1(RNAi) (Fig. 5B). S ikingly,
bo h he null mu an and he poin mu a ions wi hou he selenocys eine
p oduce esis ance o cispla in (Fig. 5C). Thus, bo h he de elopmen al
unc ion and he capaci y o p omo e cy o oxici y in he p esence o
cispla in ely on he p esence o he selenocys eine esidue.
The e o e, he elec ophilic ac i i y o hyd olyzed cispla in in he
p esence o selenocys eine migh p oduce cy o oxic SecTRAP
o ms ha a e majo d i e s o he ha m ul e ec o cispla in on
mul icellula o ganisms (Fig. 5D).
We also explo ed he ole o x -1 in sensi izing he animal o
cispla in. We ound ha x -1(s 47) con e s esis ance in ge m cells
and in soma ic cells. In e es ingly, by combining x -1(s 47) wi h
da -2 and da -16 mu a ions, he IIS pa hway is epis a ic o he x -1
ac i i y on cispla in esis ance (Fig. S1).
DISCUSSION
Despi e h ee decades o clinical use and in ense esea ch, g ea e
knowledge is necessa y o unde s and, and ul ima ely con ol, he
molecula e en s d i ing endogenous and acqui ed esis ance o
pla inum-based chemo he apy (Dil uba and Kalayda, 2016). In his
con ex , model o ganisms a e pla o ms wi h g owing in e es in
he s udy o he molecula , cellula and sys emic esponses o
chemo he apeu ic agen s, bu also o sc een o new ea men s
(Nijman, 2015). He e, we ein o ce he use o C. elegans o s udy he
complex esponse o mul icellula o ganisms o cispla in. The
unc ion o C. elegans genes in he esponse o cispla in has been
shown in di e en biological con ex s as adul su i al o ge mline
apop osis (Table S1). A e es ing some o hese con ex s, we
selec ed he body leng h o de elopmen al la ae as a eliable and
Fig. 5. x -1 null and missense mu an s a e esis an o cispla in. (A) Rep esen a ion o he x -1 gene. Blue boxes ep esen exons, and lines
ep esen in ons. The uppe line deno es he 1663 bp egion dele ed in he s 47 allele. Selenocys eine codon TGA is he hi d las esidue and is ep esen ed as
Sec. The wild- ype sequence o he las ou codons and hei co esponding amino acids a e boxed below. ce 4[U666C] and ce 5[U666STOP] Sec-de icien
x -1 mu an alleles, edi ed by CRISPR/Cas9, ca y a single subs i u ion in Sec codon (C ins ead o A o A ins ead o G) ha changes he selenocys eine o
cys eine o a p ema u e STOP codon, espec i ely. (B) Sec-de icien ce 4[U666C] and ce 5[U666STOP] alleles, in a simila manne o he x -1 null allele, cause
a mol ing-associa ed g ow h a es when ed wi h gs -1(RNAi). G aph shows he quan i ica ion o la al a es o L1 la ae ed wi h gs -1(RNAi) o con ol bac e ia
(emp y ec o ). Ba s ep esen mean and s.d. (n=50, N=2). (C) x -1(s 47) null mu an and ce 4[U666C] and ce 5[U666STOP] poin mu an s ains show
inc eased esis ance o cispla in. Body leng h quan i ica ion o synch onized L1 la ae g own on aga pla es con aining 0 and 60 µg/ml o cispla in o 48 h a 20°C.
Ba s ep esen mean and s.d. (n=50). This expe imen was pe o med in iplica e wi h simila esul s. ***P<0.001 ela i e o wild- ype wo ms in he same
condi ions by S uden ’s - es . (D) Schema ic diag am o he cy o oxic e ec p oduced by he elec ophilic ac i i y o cispla in when eac ing wi h a selonocys ein
p esen in hio edoxin educ ase 1.
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scalable sys em o e alua e he esponse o he animal o cispla in.
Nema odes a e easy o synch onize a he L1 s age, and he e o e we
can s udy homogeneous popula ions du ing la al de elopmen ,
when soma ic and ge m cells a e in ac i e p oli e a ion and
apop o ic pa hways unc ional.
The C. elegans ge mline is di e se in cell ypes, because i
con ains mi o ic cells, meio ic cells, ma u e oocy es and spe m. We
obse ed ha he e ec o cispla in on e ili y and cell cycle a es
in he mi o ic ge mline is simila o ha p oduced by o he DNA-
damaging insul s, such as ionizing o ul a iole C adia ions
(Ga ne e al., 2004), sugges ing a di ec ac ion o cispla in on DNA
ha is suppo ed by ou sou hwes e n blo s o DNA adduc s (1,2-
GpG-in as and c osslinks). This cispla in-induced DNA damage
esponse (DDR) in he C. elegans ge mline has al eady been
documen ed ( an Haa en e al., 2006; Meie e al., 2014; Honnen,
2017). In addi ion, howe e , we ound ha de ec i e spe m is a
majo cause o he educed C. elegans e ili y obse ed upon
cispla in exposu e a L4. A his s age, we hi oogenesis only
pa ially, bu ou expe imen s wi h og-2 mu an s sugges ha spe m
and oocy es a e di e en in e ms o esis ance o cispla in.
In e es ingly, cispla in is pa icula ly e icien o es icula umo s
and causes a d as ic e ec on spe ma ogenesis and spe m in ea ed
pa ien s, bu his ha m ul e ec seems o a ec p ima ily he
ch oma in and is e e sible (Bujan e al., 2013). In emales wi h
o a ian cance , cispla in also a ec s game ogenesis, p oducing a
educ ion o he o a ian ese e (Chang e al., 2015). De ailed
expe imen s need o be pe o med o gain a be e unde s anding o
he s ep a which game ogenesis is hampe ed in he p esence o
cispla in. In his sense, he he maph odi e condi ion o C. elegans is
a clea ad an age o s udy he e ec o any gi en cispla in dose on
oogenesis and spe ma ogenesis in he same o ganism.
Ou RNA-seq analyses e ealed a ansc ip omic signa u e in
he p esence o cispla in. The cispla in- esponsi e genes iden i ied
could be in ol ed in cell au onomous mechanisms (cells ha e an
indi idual esponse o cispla in up ake) o in non-cell au onomous
mechanisms ( he e ec in ce ain cells in luences dis an cells).
Gi en ha DNA is damaged by cispla in, a cell-au onomous
esponse occu s as a checkpoin mechanism o a o DNA epai
(Ga ne e al., 2004). Ne e heless, he as majo i y o genes
induced by cispla in in ou expe imen s a e ela ed o gene al s ess
esponses and he inna e immune sys em, which sugges s a
coo dina ed sys emic esponse. Acco ding o he de egula ed
genes, his esponse has DAF-16 and SKN-1 as majo d i e s and
has simila i ies o ha ob ained om nema odes exposed o IR o
UV-C (Boyd e al., 2010; G eiss e al., 2008). DAF-16 ac i a ion
posi i ely egula es a wide spec um o p ocesses, such as s ess
esis ance, inna e immuni y and me abolic adap a ion (Singh and
Aballay, 2009; Mu phy and Hu, 2013), whe eas he NRF2 C.
elegans o holog SKN-1 is an impo an ac o in de oxi ica ion
and esponse o oxida i e s ess, and i s down egula ion has been
associa ed wi h hype sensi i i y o di e en s esso agen s
(Blackwell e al., 2015).
In e es ingly, immune and s ess eac ions upon DNA damage a e
common in mammals (E molae a and Schumache , 2013), and
simila o wha we obse ed in C. elegans, cispla in induces he
nuclea ansloca ion o he DAF-16 homolog FOXO3 (Fe nandez
de Ma os e al., 2008). Human FOXO p o eins a e c ucial egula o s
o a mul i ude o cellula unc ions (Yang and Hung, 2009), bu ou
esul s encou age u he explo a ion o he manipula ion o he IIS
pa hway o con ol he cellula esponse o cispla in.
The SKN-1 human o holog NRF2 egula es he exp ession o
s ess- esponsi e genes, such as SOD and ca alases, o phase II
de oxi ica ion enzymes, such as glu a hione ans e ases (Kaspa
e al., 2009). NRF2 o e exp ession o hype ac i a ion p o okes a
di ec e ec in acquisi ion o esis ance o a wide spec um o
an icance d ugs in many cance ypes (Gañán-Gómez e al., 2013;
Kaspa e al., 2009). Acco dingly, inc eased nuclea NRF2
exp ession has been shown in cispla in- esis an human bladde
cance samples (Hayden e al., 2014). On he con a y, inhibi ion o
NRF2 sensi izes cispla in- esis an A549 cells (Hou e al., 2015).
These obse a ions a e conco dan wi h ou indings in C. elegans,
sugges ing ha di ec o indi ec a ge ing o NRF2 should be
explo ed o cispla in-combined he apeu ic in e en ions.
Ou ansc ip omic analyses unco e ed he up egula ion o egl-1
and ced-13, which a e ela ed o apop osis. Using a egl-1p::GFP
epo e , we ound ec opic exp ession in soma ic cells. The apop o ic
pa hway in pos emb yonic soma ic cells is no well desc ibed and
migh be di e en in each soma ic cell ype. In he canonical iew o
he DNA damage-induced apop o ic pa hway, exp ession o he
e ec o EGL-1 ma ks cells des ined o die (Nehme and Con ad ,
2008), and cep-1/p53 is ups eam (Schumache e al., 2005a).
Howe e , egl-1p::GFP is s ill o e exp essed in cep-1/p53 mu an s
exposed o cispla in, indica ing ha inhibi ion o he DNA damage-
induced apop o ic pa hway does no comple ely p o ec he animal
om cispla in oxici y. Likewise, blockade o apop osis in adul
wo ms h ough cep-1/p53 inhibi ion does no ha e any e ec on
cispla in sensi i i y (Hemmingsson e al., 2010). Thus, inhibi ion o
he DNA damage-induced apop o ic pa hway is no an e icien
s a egy o animals o hampe he global e ec o cispla in.
None heless, exp ession o cep-1/p53 seems o be impo an o he
ull esponse o cispla in, and p53 is equi ed o he cy o oxic e ec
o cispla in in human glioblas oma cells (Pa k e al., 2006).
Su p isingly, we ound ha ced-13 up egula ion migh ha e a
p o ec i e ole, because ced-13 mu an s a e sensi i e o cispla in.
Mu an s o ced-3, a gene ha is gene ically downs eam, a e also
sensi i e o cispla in. This esul i s wi h he ole o ced-13 in he
mi ochond ial in insic apop o ic pa hway ha ex ends C. elegans
longe i y (Yee e al., 2014). I is possible ha pos mi o ic cells,
which a e i eplaceable, ac i a e ced-13 ac i i y o p o ec hemsel es
om cispla in. The ole o ced-13 migh be dependen on he insul
le els o a y in dis inc cell ypes, because o e exp ession o ced-13
in he soma induces cell dea h o soma ic cells ha no mally su i e
(Schumache e al., 2005a). In conco dance wi h a p o ec i e ole o
ced-13 and ced-3, he ac i i y o hese genes appea s o p o ec
dopamine gic neu ons om he oxici y o an oxida i e s ess-
inducing d ug (O enbu ge e al., 2018).
Thio edoxin sys em p o eins a e key playe s in many impo an
cellula p ocesses, including he main enance o edox homeos asis
(A né , 2009; Lu and Holmg en, 2014). Rega ding he in luence o
hio edoxins in he cellula esponse o cispla in, in mammalian
cells i has been shown ha a delica e balance exis s be ween he
cy o oxic e ec o he di ec in e ac ion wi h cispla in and he
p o ec i e e ec , main aining he edox equilib ium (Anes ål e al.,
2008; Cebula e al., 2015). S udies in cell lines sugges ha
cispla in cy o oxici y is p omo ed by a di ec in e ac ion be ween
cispla in and selenocys eine amino acids, leading o he consequen
o ma ion o SecTRAPs (Anes ål e al., 2008). We con i med he
cispla in-induced cy o oxici y o TRXR-1 and, gene a ing wo
dis inc missense mu a ions by CRISPR/Cas9, we demons a ed
ha his cy o oxici y elies on he p esence o a single
selenocys ein (Sec) a he C- e minal o he p o ein. In e es ingly,
he p esence o Cys ins ead o Sec in TRXR1 can be modula ed by
he p esence o he mic onu ien selenium in he die o a s
(Lu e al., 2009).
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The mul i ac o ial na u e o cispla in esis ance hampe s he
inding o a unique solu ion o o e come he esis ance o umo s o
cispla in. Howe e , he e a e many pa allels be ween he esponse
o C. elegans and mammals o a cispla in ea men ha would
acili a e he s udy in nema odes o pa icula gene ic, me abolic and
en i onmen al ac o s in luencing he esis ance o cispla in. This
in o ma ion would be o g ea help o iden i y p edic i e bioma ke s
and in es iga e new d ugs o ha e mo e e ec i e and pe sonalized
cispla in-based he apies.
MATERIALS AND METHODS
Nema ode s ains and gene al me hods
Caeno habdi is elegans s ains we e cul u ed and main ained using s anda d
p ocedu es (Po a-de-la-Ri a e al., 2012; S ie nagle, 2006). The N2
(B is ol) s ain was used as wild- ype in all expe imen s, and he ollowing
alleles and ansgenic s ains we e used in his s udy: BS553, og-2(oz40) V;
XF132, polh-1(i 31) III; CB1370, da -2(e1370) III; TJ1, cep-1(gk138) I;
CE1255, cep-1(ep347) I; FX536, ced-13( m536) X; MD792, ced-13(s 32)
X; VB1414, x -1(s 47) IV; CER170, x -1(ce 4[U666C])IV; CER171,
x -1(ce 5[U666s op])IV; CF1038, da -16(mu86) I; TJ356, zIs356
[da -16p::da -16a/b::GFP + ol-6(su1006)] IV; WS1973, opIs56 [egl-
1p::2xNLS::GFP]; CER192, cep-1(gk138) I, opIs56(egl-1p::GFP); and
CL2166, d Is19 [pAF15(gs -4p::GFP::NLS)] III.
CRISPR/Cas9
To gene a e he x -1(ce 4[U666C])and x -1(ce 5[U666S op])poin
mu a ion alleles, we a ge ed a si e nea he selenocys eine codon wi h
CRISPR/Cas9. To make he sgRNA exp ession plasmid, we cloned he
annealed oligonucleo ides in o BsaI-diges ed U6::sgRNA pMB70
(Waaije s e al., 2013). Repai empla es con aining desi ed modi ica ions
we e designed by Gibson Assembly Cloning Ki (New England BioLabs)
and cloned in o he pBSK plasmid (Addgene). Oligonucleo ide sequences
used o gene a e he 5′and 3′1.5 kb o e lapping agmen s a e a ailable on
eques . Bo h sgRNA and epai empla e we e e i ied by sequencing using
T4 and M13 p ime s, espec i ely. Young adul wild- ype animals we e
injec ed wi h 30 ng/μl o sgRNA, 100 ng/μl o epai empla e, 30 ng/μlo
Pe -3::Cas9 ec o (F iedland e al., 2013) and 2.5 ng/μlo Pmyo-2::
dToma o. Single luo escen p ogeny we e isola ed and sc eened o he
p esence o he mu a ion by PCR. We inally es ablished and alida ed
homozygous mu an lines by PCR and sequencing.
RNA-sequencing analyses
A mixed popula ion o wo ms ep esen ing all s ages and g own in con ol
condi ions was exposed o 60 µg/ml o cispla in. A e 24 h a 20°C,
cispla in- ea ed wo ms and un ea ed con ol animals we e washed wi h M9
bu e o emo e bac e ia, and o al RNA was ex ac ed using he TRIzol
me hod. To al mRNA was subsequen ly en iched using he mi Vana
miRNA isola ion ki (Ambion) ollowed by poly-A cap u e. Lib a y
cons uc ion and Illumina’s HiSeq 2000 echnology sequencing was
pe o med ollowing he manu ac u e ’s ins uc ions. App oxima ely 12
million eads (100 bp leng h) o each sample we e p ocessed and aligned
using TopHa so wa e o he C. elegans e e ence genome, e sion
WBcel235.74, o p oduce BAM iles [Gene Exp ession Omnibus (GEO)
da abase e e ence GSE111654]. These BAM iles we e analyzed wi h he
SeqSol e NGS so wa e (In eg omics, S.L.), using a alse disco e y a e o
0.05 and il e ing eads displaying mul iple mapping si es. SeqSol e uses
Cu links and Cu di (T apnell e al., 2012) p og ams o pe o m
di e en ial gene exp ession analyses be ween samples (P<0.005).
Exp ession alues we e no malized in FPKM ( agmen s pe kilobase o
exon pe million agmen s mapped). Da ase s o up- and down egula ed
genes we e gene a ed, compa ing he di e en ial gene exp ession analyses
o wo independen eplica es (P<0.01 and P<0.05).
Cispla in assay du ing la al de elopmen
A synch onized popula ion o L1-a es ed la ae was cul u ed on NGM
pla es con aining esh OP50 and 0-200 µg/ml o cispla in (Sigma). The
body leng h o ≥50 wo ms o each condi ion was measu ed a 48, 72 and
96 h a 20°C a he s e eomic oscope using NIS-Elemen s 3.2 imaging
sys em. The subsequen analyses we e pe o med wi h 60 µg/ml o cispla in
in he same condi ions excep o assays con aining da -2(e1370) s ain,
which we e pe o med a 15°C, measu ing he body leng h a e 4 days o
incuba ion o a oid empe a u e- ela ed de elopmen al delay. Each assay
was done in duplica e, and a leas wo biological eplica es we e pe o med.
Nonpa ame ic S uden ’s - es (G aphPad P ism 5) was used o de e mine
he signi icance o di e ences in he mean.
Analysis o he e ec o cispla in in he ge mline
To analyze he e ec o cispla in on he ge mline, a synch onized popula ion
o L4-s age animals g own in s anda d condi ions (39 h a 20°C) was
ans e ed on o NGM pla es con aining 60 µg/ml cispla in. A e 24 h o
incuba ion a 20°C, wo ms we e washed in PBS and ge mlines dissec ed in
wo ms anes he ized wi h 3 mM o le amisol in PBS, ixed wi h 4%
pa a o maldehyde and s ained wi h DAPI (0.6 µg/ml). These s ained gonads
we e pho og aphed using a Nikon ECLIPSE TI-s in e ed mic oscope. Fo
ge m cell quan i ica ion in he p oli e a i e egion, he o al numbe o cells
in a single z-s ack wi hin 50 µm o he dis al end o he gonad was coun ed.
A leas 15 ge mlines we e coun ed o each expe imen .
B ood size and un e ilized oocy es
To pe o m he sel - e iliza ion assays, a synch onized popula ion o L3-s age
wo ms was exposed o NGM pla es, wi h addi ion o esh OP50 and 0, 60 o
100 µg/ml o cispla in o 24 h. A o al o 12 wo ms om each condi ion we e
selec ed on o esh OP50 pla es, coun ing he p ogeny and he numbe o
oocy es laid a e 3 days. A simila p o ocol was pe o med o c ossing
assays, whe e we used ei he a male-en iched popula ion in he case o N2, o
he male– emale popula ion o he og-2(oz40) allele. A e 24 h o exposu e
o cispla in, a o al o 12 gene ic c osses we e pe o med o each condi ion,
placing one he maph odi e, o emale in he case o og-2(oz40), and ou
males on esh OP50 pla es, le ing hem ma e and lay p ogeny o 3 days. This
expe imen was pe o med in duplica e. A nonpa ame ic S uden ’s - es was
used o de e mine he signi icance o di e ences in he mean.
In i o in acellula localiza ion o DAF-16
To de e mine he subcellula localiza ion o DAF-16, a synch onized L2-
s age popula ion o wo ms ca ying he DAF-16::GFP ansgene (TJ356)
was ans e ed o M9 bu e con aining 0, 90 o 180 µg/ml o cispla in o
5 h a 20°C. Then, he wo ms we e washed in M9 and moun ed on a
mic oscope slide con aining a 2% aga pad, using a d op o 3 mM
le amisole o anes he ize hem. We quan i ied he DAF-16 subcellula
localiza ion by conside ing as ‘nuclea ’wo ms showing a mainly nuclea
GFP accumula ion along he whole body, as shown in Fig. 3B. A o al o 50
wo ms we e obse ed in each one o h ee independen eplica es.
egl-p::GFP exp ession assays
To de e mine ec opic egl-1 exp ession, a synch onized L1-s age popula ion
o egl-1p::GFP (WS1973) ansgenic nema odes was g own on NGM pla es
con aining esh OP50 and 60 μg/ml cispla in pla es o 24 h a 20°C. Fo in
i o obse a ion, wo ms we e eco e ed wi h M9 bu e , washed and
moun ed on a mic oscope slide con aining a 2% aga pad, using a d op o
3 mM le amisole o anes he ize hem. We used a Nikon ECLIPSE TI-s
in e ed mic oscope o quan i y he numbe o cells showing an ec opic GFP
signal. These expe imen s we e pe o med h ee independen imes.
RNAi and in i o gs -4 exp ession assay
da -16 and skn-1 RNAi clones used in his s udy we e ob ained om he
ORFeome lib a y (Rual e al., 2004). RNAi by eeding was pe o med in
s anda d condi ions, using NGM pla es supplemen ed wi h 50 μg/μl
ampicillin, 12.5 μg/μl e acycline and 3 mM IPTG. To analyze gs -4
induc ion, a synch onized L1-s age popula ion o gs -4p::GFP epo e
s ain was g own on pla es con aining he co esponding RNAi clone o
24 h a 20°C. Then, wo ms we e ans e ed o new RNAi pla es including 0
o 60 μg/ml cispla in o 24 h. Then, wo ms we e eco e ed wi h M9 bu e ,
washed and moun ed on a mic oscope slide con aining a 2% aga pad, using
a d op o 3 mM le amisole o anes he ize hem. A Nikon ECLIPSE TI-s
in e ed mic oscope was used o in i o obse a ion, and image analysis
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RESEARCH ARTICLE Disease Models & Mechanisms (2018) 11, dmm033506. doi:10.1242/dmm.033506
Disease Models & Mechanisms