RESEARCH ARTICLE
Skipped BSCL2 T ansc ip in Celia’s
Encephalopa hy (PELD): New Insigh s on
Fa y Acids In ol emen , Senescence and
Adipogenesis
So ía Sánchez-Iglesias
1
, Alexande Un uh-Pinhei o
1
, C is ina Guillín-Ama elle
1
,
Blanca González-Méndez
1
, Alejand o Ruiz-Riquelme
1
, Blanca Le icia Rod íguez-Cañe e
1
,
Sil ia Rod íguez-Ga cía
1
, Enca nación Guillén-Na a o
2
, Rosa io Domingo-Jiménez
3
,
Da id A aújo-Vila
1
*
1Thy oid and Me abolic Diseases Uni (U.E.T.eM.), Depa men o Medicine, Cen e o Resea ch in
Molecula Medicine and Ch onic Diseases (CIMUS)-IDIS, Uni e si y o San iago de Compos ela, San iago
de Compos ela, Spain, 2Uni o Medical Gene ics and Dysmo phology, Di ision o Pedia ics, Hospi al
Clínico Uni e si a io Vi gen de la A ixaca, Mu cia, Spain, 3Sec ion o Neu opedia ics, Di ision o
Pedia ics, Hospi al Clínico Uni e si a io Vi gen de la A ixaca, Mu cia, Spain
*[email p o ec ed]
Abs ac
Objec i e
PELD (P og essi e Encephalopa hy wi h o wi hou Lipodys ophy o Celia’s Encephalopa-
hy) is a a al and a e neu odegene a i e synd ome associa ed wi h he BSCL2 mu a ion
c.985C>T, ha esul s in an abe an ansc ip wi hou he exon 7 (Celia seipin). The aim o
his s udy was o e alua e bo h he p ocess o cellula senescence and he e ec o unsa u-
a ed a y acids on p eadipocy es om a homozygous c.985C>T pa ien . Also, he ole o
abe an seipin iso o m on adipogenesis was s udied in adipose-de i ed human mesenchy-
mal s em cells.
Ma e ial and me hods
Cellula senescence was e alua ed using β-galac osidase s aining o p eadipocy es
ob ained om a homozygous c.985C>T pa ien . Mo eo e , hese cells we e cul u ed du ing
24 hou s wi h In alipid, a soybean oil-based comme cial lipid emulsion. The exp ession o
he di e en BSCL2 ansc ip s was measu ed by qPCR. Adipose-de i ed human mesen-
chymal s em cells we e di e en ia ed o a a lineage using S emPRO adipogenesis ki , and
he exp ession o BSCL2 ansc ip s and se e al adipogenesis- ela ed genes was mea-
su ed by qPCR.
Resul s
he ea men o p eadipocy es wi h unsa u a ed a y acids signi ican ly educed he exp es-
sion o he BSCL2 ansc ip wi hou exon 7 by 34 o 63%. On he o he hand, a leas in p e-
adipocy es, his mu a ion does no dis u b cellula senescence a e. Finally, du ing
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 1/13
a11111
OPEN ACCESS
Ci a ion: Sánchez-Iglesias S, Un uh-Pinhei o A,
Guillín-Ama elle C, González-Méndez B, Ruiz-
Riquelme A, Rod íguez-Cañe e BL, e al. (2016)
Skipped BSCL2 T ansc ip in Celia’s Encephalopa hy
(PELD): New Insigh s on Fa y Acids In ol emen ,
Senescence and Adipogenesis. PLoS ONE 11(7):
e0158874. doi:10.1371/jou nal.pone.0158874
Edi o : Miguel López, Uni e si y o San iago de
Compos ela School o Medicine—CIMUS, SPAIN
Recei ed: Ap il 12, 2016
Accep ed: June 23, 2016
Published: July 8, 2016
Copy igh : © 2016 Sánchez-Iglesias e al. 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, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any
medium, p o ided he o iginal au ho and sou ce a e
c edi ed.
Da a A ailabili y S a emen : All ele an da a a e
wi hin he pape .
Funding: This wo k was suppo ed by he Ins i u o
de Salud Ca los III and he Eu opean Regional
De elopmen Fund, FEDER (g an s numbe PI10/
02873 and PI13/00314, h p://www.isciii.es/), ISCIII/
PI13/00314/Co inanciado FEDER and by he
Conselle ía de Indus ia, Xun a de Galicia (g an
numbe 10PXIB208013PR, h p://www.xun a.es/).
SRG was awa ded a Resea ch Fellowship, g an ed
by he Asociación Española de Familia es y
adipocy e di e en ia ion o adipose-de i ed human mesenchymal s em cells, he exp es-
sion o adipogenic genes (PPARG,LPIN1, and LPL) inc eased signi ican ly o e 14 days,
and no ewo hy is ha he BSCL2 ansc ip wi hou exon 7 was di e en ially exp essed by
332 o 723% when compa ed o day 0, sugges ing an unde lying ole in adipogenesis.
Conclusions
ou esul s sugges ha Celia seipin is p obably playing an unde es ima ed ole in adipocy e
ma u a ion, bu no in senescence, and i s exp ession can be modi ied by exogenous ac-
o s as a y acids.
In oduc ion
Seipin is a p o ein whose unc ion has no been ye ully elucida ed. Mu a ions in BSCL2 gene
a e ela ed o ype 2 Be a dinelli-Seip congeni al lipodys ophy ( ype 2 CGL) [1] and also wi h
a ious congeni al neu opa hies [2]. Seipin is a memb ane p o ein o he endoplasmic e iculum
(ER). P edic i e algo i hms and expe imen al da a sugges ha i consis s o wo ansmemb ane
domains, a highly conse ed in aluminal loop, and wo N- e minal and C- e minal domains in
cy oplasm [3].
Unde na u al condi ions, BSCL2 gene encodes mainly h ee ansc ip s o 462 (BSCL2-003,
ENST00000360796; CCDS44627), 398 (BSCL2-004/005/006, ENST00000421906; ENST00000407022;
ENST00000403550; CCDS8031) and 287 (BSCL2-008, ENST00000278893; CCDS55769) amino
acids espec i ely. The i s ansc ip desc ibed [1]wasBSCL2-004/005/006.BSCL2-003 is iden i-
cal o he i s one excep o a N- e minal ex ension o 64 amino acids encoded by exon 1. The
287-amino acid sho ansc ip ea u es skipping o exon 7, which esul s in a change in he
eading ame, so ha he esul ing p o ein is comple ely di e en om he o he wo ones in he
amino acid s e ch encoded om exon 6 o exon 10.
The unc ion o seipin is s ill being in es iga ed, howe e , i s ela ionship wi h adipogenesis,
wi h he genesis o lipid d ople s and he egula ion o he me abolism o phospholipids and
iacylglyce ides has been es ablished [4–10]. I was also sugges ed ha seipin migh play an
impo an ole in he ne ous sys em [11–15].
Type 2 CGL synd ome is a a e au osomal ecessi e disease cha ac e ized by an almos o al
lack o adipose issue in he body. In addi ion o he lack o adipose issue, o he clinical ea-
u es a e insulin esis ance, li e s ea osis and hype iglyce idemia, which can e ol e in o dia-
be es melli us and ea ly ci hosis. Type 2 CGL is also cha ac e ized by a cogni i e delay o
a iable se e i y [16]. Mo eo e , BSCL2-associa ed neu opa hies a e au osomal dominan dis-
o de s a ec ing uppe and/o low mo o neu on wi hou a ec ing li e expec ancy.
We ha e p e iously epo ed a new seipin-associa ed neu odegene a i e synd ome (PELD,
P og essi e Encephalopa hy wi h/wi hou Lipodys ophy o Celia’s Encephalopa hy, MIM:
#615924) [17] due o a c.985C>T mu a ion in BSCL2 gene. This mu a ion gi es ise o an al e -
na i e splicing leading o skipping o exon 7 and a eading ame shi esul ing in a new abe an
p o ein, he ea e called Celia seipin [17], simila o BSCL2-008 ansc ip . Pa ien s su e ing
PELD die p ema u ely be o e age 9 as a consequence o a se e e and p og essi e encephalopa hy.
No ewo hy, homozygous cases ha dly show lipodys ophic ea u es while compound he e ozy-
gous cases ha e a mixed pheno ype, neu odegene a i e and lipodys ophic.
Despi e i s ecessi e pa e n o inhe i ance, we ha e p oposed a oxic gain o unc ion mech-
anism, so ha Celia seipin accumula ion in he ER o neu ons migh induce he un olded p o-
ein esponse (UPR) esponsible o cellula damage and apop osis [17,18].
Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 2/13
A ec ados de Lipodis o ias (AELIP, h p://www.aelip.
o g/). The unde s had no ole in s udy design, da a
collec ion and analysis, decision o publish, o
p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed
ha no compe ing in e es s exis .
A ele an ac in he na u al his o y o his neu odegene a i e condi ion is ha neu ological
clinical signs do no appea immedia ely. Thus, a ec ed child en a e bo n heal hy om a neu-
ological poin o iew. The homozygous pa ien s mani es psychomo o delay be ween 12 and
24 mon hs o age and neu ological in olu ion becomes e iden a e 3 yea s. In compound he -
e ozygo es, lipodys ophy was e iden om he i s mon h o li e, while he neu ological na u-
al his o y was simila o he homozygous cases. All pa ien s died be ween 7–8 yea s, excep he
younges one, who is s ill, a 6.8 yea s o age, in he ea ly s ages o he neu ological condi ion.
Gi en his s iking disease e olu ion, we wonde ed whe he c.985C>T mu a ion could in e -
e e in he p ocess o cellula senescence. In his line, in i o s udies wi h yeas , ha e shown he
impo ance o a co ec lipolysis o he p og ession o he cell cycle [19], and mu a ions in
FLD1, he o hologous gene o BSCL2, dec eases lipolysis [10], sugges ing a possible molecula
ela ionship wi h cellula senescence.
On he o he hand, all hese pa ien s, bo h homozygous and compound he e ozygous, p e-
sen ed, a leas du ing he i s mon hs o li e, e y high le els o plasma iglyce ides. I is well
known [20] ha he con ibu ion o essen ial a y acids is key o a no mal de elopmen o he
ne ous sys em in humans, mainly up o 3 yea s o age, and, as al eady men ioned, seipin plays
a c ucial ole in he o ma ion o lipid d ople s and in he syn hesis o phospholipids [4,5,7,
21]. The e o e, ano he hypo hesis o his s udy is ha he con ibu ion o a y acids in he die
migh in luence he disease e olu ion o his neu odegene a i e p ocess.
In addi ion, p e ious s udies o ou g oup sugges ha BSCL2-008 ansc ip migh play a
posi i e ole in he main enance o adipogenesis, conside ing ha clinical ea u es o ype 2
CGL e e o a e no p esen in homozygous subjec s o he c.985C>T mu a ion [17]. Since
no ela ion be ween sho human BSCL2 ansc ip and adipogenesis has been es ablished
be o e, his led us o s udy he in ol emen o BSCL2 ansc ip s du ing he di e en ia ion o
adipose-de i ed human mesenchymal s em cells (ADSCs).
Ma e ials and Me hods
This s udy was app o ed by he E hics Re iew Panel o Xun a de Galicia, and ca ied ou
acco ding o he e hical guidelines o he Helsinki Decla a ion. W i en in o med consen was
ob ained om he pa en s o he pa icipan s included in he s udy.
Adipose issue biopsies and cell cul u e
A small sample o subcu aneous adipose issue o he abdominal a ea was ob ained om he
index case (homozygous o c.985C>T mu a ion), a 6 yea s o age. A con ol adipose issue
sample was ob ained, in acco dance wi h e hical egula ions o cu en Spanish legisla ion,
om a 6-yea -old con ol boy who unde wen p og ammed abdominal su ge y o c yp o chi-
dism. The collec ed explan s we e p ocessed immedia ely ollowing sampling. Small pieces o
adipose issue we e insed wi h PBS and placed on a 60 mm dish (BD Falcon
TM
; Mississauga,
ON, Canada) con aining Dulbecco’s modi ied Eagle’s medium (DMEM; Sigma-Ald ich,
S . Louis, MO, USA) plus 30% e al bo ine se um (FBS) and gen amicin 50 μg/mL (Sigma-
Ald ich), and incuba ed a 37°C wi h 5% CO
2
in a Wa e -Jacke CO
2
incuba o (NuAi e, Plym-
ou h, MN, USA). A e one week, p eadipocy es ha had mig a ed ou o he explan s we e ec-
ognized by he p esence o small lipid d ople s wi hin he ib oblas -like cells. The explan s
issues we e hen emo ed om he dishes and he cells we e u he cul u ed wi h esh
medium un il a 70% con luen monolaye was ob ained. A e wa ds, hese p eadipocy es we e
ipsinized (T ypLE™Exp ess S able T ypsin-like Enzyme wi h Phenol Red; Gibco Li e Tech-
nologies; Ca lsbad, CA, USA) and cul u ed on 100 mm dishes in DMEM con aining 10% FBS
and penicillin-s ep omycin 1% (Sigma-Ald ich). Comme cially a ailable ADSCs we e
Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 3/13
pu chased om In i ogen (S emP o human adipose-de i ed s em cells, In i ogen, Ca lsbad,
CA, USA). In i ogen isola ed hese ADSCs om he abdominal egion o a 39-yea -old emale
dono using lipoaspi a e, he cells we e hen expanded o one passage p io o c yop ese a-
ion and assayed o pu i y using low cy ome ic analysis o cell su ace an igen exp ession.
ADSCs we e main ained in issue cul u e g ade uncoa ed Pe i dishes (BD Falcon
TM
) in com-
ple e MesenPRO RS™medium (In i ogen) supplemen ed wi h 1x Glu aMAX™-I (Gibco) and
gen amicin 50 μg/mL, and incuba ed a 37°C wi h 5% CO
2
in a Wa e -Jacke CO
2
incuba o
(NuAi e). Cul u e medium was changed e e y h ee days, and he cells we e spli a 80% con-
luence. ADSCs we e used a low-passage 3 o all expe imen s.
T ea men wi h a y acids
P eadipocy es we e cul u ed on 35 mm mul iwell dishes (6-well pla es) in DMEM con aining
10% FBS and penicillin-s ep omycin 1% supplemen ed du ing 24 hou s wi h 83.3 mg/dL
In alipid (200 mg/mL, F esenius Kabi, Uppsala, Sweden), a soybean oil-based comme cial
lipid emulsion ha is en iched p edominan ly wi h polyunsa u a ed a y acids (PUFA: 61%);
speci ically, he a y acid composi ion o In alipid is 54.7% linoleic, 20.9% oleic, 11% palmi ic,
6.6% alpha-linolenic and 3.9% s ea ic [22]. The dose was chosen in ag eemen o ha used in
human pa en e al nu i ion assuming a dis ibu ion olume o 5 L.
Senescence associa ed β-galac osidase s aining
Exp ession o pH-dependen senescence associa ed β-galac osidase (SA-β-gal) ac i i y was ana-
lyzed simul aneously in di e en passages o con ol and index case p eadipocy es using he
SA-β-gal s aining ki (Cell Signaling Technology, Bos on, MA) [23]. Cells we e cul u ed in
24-well pla es (6.5 mm mul iwell dishes) du ing 24 hou s, washed in PBS, ixed o 15 minu es
a oom empe a u e in 2% o maldehyde/0.2% glu a aldehyde, washed again, and incuba ed
o e nigh a 37°C (wi hou CO
2
) wi h esh s aining solu ion: 1 mg/mL o 5-b omo-4-chlo o-
3-indolyl β-D-galac oside (X-Gal) in dime hyl o mamide, 40 mM ci ic acid/sodium phos-
pha e, pH 6.0, 5 mM po assium e ocyanide, 5 mM po assium e icyanide, 150 mM NaCl, 2
mM MgCl
2
. Cell nuclei we e s ained wi h 5 μl/well o 50 μM luo och ome bisbenzamide
(Hoechs 33258; San a C uz Bio echnology, San a C uz, CA, USA) du ing 40 minu es a oom
empe a u e. Cells we e isualized wi h an Olympus IX51 mic oscope (Olympus Co po a ion,
Tokyo, Japan) by b igh ield mic oscopy and pho og aphed wi h an Olympus DP72 digi al
came a using he cellSens so wa e (Olympus Co po a ion). Quan i ica ion was pe o med
wi h ImageJ so wa e (Na ional Ins i u e o Men al Heal h) in iplica e, aking 4 andom non-
o e lapping ields o iew o each sample.
Adipogenic di e en ia ion and Oil Red O s aining
To induce ADSCs adipogenic di e en ia ion, cul u e medium was swi ched o a comme cially
a ailable di e en ia ion media ki , S emPRO adipogenesis ki (In i ogen). Cul u e medium
was changed e e y h ee days. A day 14 a e induc ion o adipogenic di e en ia ion, ADSCs
we e s ained wi h Oil Red O (Sigma-Ald ich) o e eal ully di e en ia ed cells. Cells we e
washed wi h PBS and ixed in 10% o malin in iso onic bu e o 1 hou a oom empe a u e.
A e insing h ee imes wi h double dis illed H
2
O o emo e esidual o malin, lipid acuoles
we e s ained wi h 0.6% Oil Red O (weigh / olume) in 60% isop opanol o 1 hou a 22°C and
washed h ee imes wi h double dis illed H
2
O. Following Oil Red O s aining, cells we e inally
isualized wi h an Olympus IX51 mic oscope (Olympus Co po a ion) and pho og aphed wi h
an Olympus DP72 digi al came a using he so wa e cellSens (Olympus Co po a ion).
Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 4/13
RNA ex ac ion and e o ansc ip ion
To al RNA was ex ac ed om p eadipocy es and ADSCs using T izol (In i ogen) as pe he
manu ac u e ’s ins uc ions. RNA was e e se- ansc ibed using M-MLV e e se ansc ip ase
(In i ogen) as p e iously desc ibed [24].
cDNA PCR
BSCL2 cDNA was ampli ied wi h p ime s designed wi h he P ime 3Plus so wa e (h p://
www.bioin o ma ics.nl/cgi-bin/p ime 3plus/p ime 3plus.cgi) ( o wa d: 5’-CAGATGCTGGA
CACACTGGT-3’, e e se: 5’-ATCACTGGCCTCAGGCTCTA-3’). PCR condi ions we e as
ollows: 5 min a 95°C ollowed by 40 cycles, each consis ing o 30 s a 95°C, 30 s a 59°C, and 1
min a 72°C, hen a 10 min inal elonga ion a 72°C. Ampli ica ion agmen s ob ained we e
sepa a ed ou by aga ose gel elec opho esis (1%).
Real- ime RT-PCR
Speci ic p ime s and p obes designed by Uni e sal P obeLib a y (Roche Diagnos ics, San
Cuga del Valles, Spain) we e used in a Ligh Cycle 2.0 (Roche Diagnos ics) o de e mine spe-
ci ically exp ession o adipogenic genes PPARG,LPIN1 and LPL, and wo dis inc BSCL2 an-
sc ip s on days 0, 1, 2, 3, 4, 5, 6, 7, 8 and 14 o adipogenic di e en ia ion. The p obe #73
(TCCTCAGC) used wi h o wa d: 5’-TGCGCCTTCATAGGTGTTG-3’, and e e se p ime :
5’-ACCCACTGCATGTAGCTGAA-3’, hyb idizes wi h exon 7 o BSCL2 gen (amplicon 74
nucleo ides), while he e e se p ime : 5’-AGCGATCATTGAGATCCACA-3’, used wi h he
p obe #42 (CATCCAGC) and o wa d p ime : 5’-TTTTCGGATGTTAACCTGAGC-3’, exclu-
si ely hyb idizes wi h he union o exon 6 and exon 8 (amplicon 96 nucleo ides). Real- ime
RT-PCR condi ions a e a ailable upon eques . Resul s we e no malized o he RNA polyme -
ase II o 18s RNA genes using he 2-ΔΔ CT me hod [25].
S a is ical Analysis
The s a is ical signi icance was de e mined using non-pa ame ic K uskal-Wallis es , ollowed
by a Mann-Whi ney U pos -hoc wi h Bon e oni co ec ion. Senescence assay was pe o med
in iplica e, aking pho og aphs o 4 andom ields pe well, and eal- ime RT-PCR analyses in
duplica e, n = 4. Da a a e p esen ed as mean ± SD wi h s a is ical signi icance se a P<0.05. All
s a is ical analyses we e pe o med using SPSS o PC ( elease 22.0; SPSS, Chicago, IL, USA).
Resul s
Exon 7 skipping con i ma ion
P eadipocy es om index case and con ol we e cul u ed and analyzed by PCR o s udy he
exp ession o seipin. As shown in Fig 1A, con ol cells showed a single band in elec opho esis,
while he index case samples showed a second band o lowe molecula weigh , co esponding
o he exon 7 skipped Celia ansc ip , in ag eemen wi h ou p e ious s udy [17]. Mo eo e ,
his elec opho e ic di e en ial pa e n emained iden ical a all passages. Simila ly, a e ea -
men wi h In alipid, he elec opho e ic pa e n did no change in he di e en passages o
bo h con ol and index case samples (Fig 1B).
Exp ession o seipin ansc ip s
The basal exp ession o he ansc ip s con aining exon 7 was much highe (72- old, passages
a e age) han ha o he exon 7-skipped ansc ip in con ol cells, while his was no so in
Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 5/13
cells om he index case, in which he exp ession o he wo ansc ip s was in a simila o de
(Table 1A). A basal condi ions and as expec ed, he ela i e exp ession o he la ge ansc ip s
was educed in p eadipocy es wi h he c.985C>TBSCL2 Celia mu a ion (13%) as compa ed
wi h con ol cells, howe e , he exp ession o he skipped ansc ip in mu an cells was a ound
10-17- old highe han ha was obse ed in con ol cells (Table 1A).
Rega ding eal ime RT-PCR s udies o con ol and index case p eadipocy es, no signi ican
di e ences we e de ec ed in he ela i e exp ession o he ansc ip s con aining exon 7 a e
Fig 1. Ampli ica ion esul s o he splicing egion. Resul s o he ampli ica ion o he 573 bp egion o cDNA om p eadipocy es om he index case and
con ol subjec in di e en passages wi hou In alipid ea men (a) o in he p esence o In alipid (b). Only samples om he index case show he p esence
o he 431 base pai -long addi ional band which once sequenced con i med he skipping o exon 7.
doi:10.1371/jou nal.pone.0158874.g001
Table 1. Rela i e exp ession o BSCL2 ansc ip s.
A) Rela i e exp ession o BSCL2 ansc ip s a basal condi ions
CONTROL INDEX CASE
Wi h exon 7 Wi hou exon 7 Wi h exon 7 Wi hou exon 7
P8 1.034±0.275 0.012±0.004 *0.136±0.035
§
0.120±0.026
†
P12 1.036±0.302 0.017±0.005 *0.164±0.028
§
0.282±0.062
†‡#
P15 1.109±0.321 0.016±0.006 *0.172±0.051
§
0.195±0.059
†
B) Rela i e exp ession o BSCL2 ansc ip s a e In alipid ea men
CONTROL INDEX CASE
Wi h exon 7 Wi hou exon 7 Wi h exon 7 Wi hou exon 7
P8 1.096±0.214 0.013±0.002 *0.101±0.017
§
0.079±0.011
†
P12 1.412±0.291
‡
0.012±0.002 *0.142±0.030
§
0.102±0.029
†
P15 1.286±0.236 0.010±0.002 *0.205±0.036
§‡
0.103±0.020
†‡#
Rela i e exp ession o BSCL2 ansc ip s e e ed o BSCL2 ansc ip s wi h exon 7 in con ol (A) a basal condi ions, and (B) a e In alipid ea men :
skipped ansc ip e e ed o BSCL2 ansc ip s con aining he exon 7 in con ol
*:P<0.05; skipped ansc ip e e ed o BSCL2 ansc ip s con aining he exon 7 in index case
#: P<0.05; ansc ip s wi h exon 7 in index case e e ed o BSCL2 ansc ip s con aining he exon 7 in con ol
§: P<0.05; skipped ansc ip in index case e e ed o BSCL2 skipped ansc ip in con ol
†:P<0.05
‡:P<0.05 s. P8. P: passage. All samples we e analyzed in duplica e, n = 4. Resul s we e no malized o he RNA polyme ase II gene.
doi:10.1371/jou nal.pone.0158874. 001
Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 6/13
In alipid ea men no in he con ol nei he in he index case cells (Fig 2A,Table 1B), while
he e we e signi ican di e ences in he ela i e exp ession o he skipped ansc ip in he
index case p eadipocy es (Fig 2B,Table 1B). Thus, an unsa u a ed a y acid en iched medium
signi ican ly educed (-48% on a e age o he di e en passages) he exp ession o he an-
sc ip wi hou exon 7.
Fig 2. Rela i e exp ession and pe cen age o change in exp ession o BSCL2 ansc ip s a e In alipid
ea men . (a) Pe cen age o change e e ed o he ela i e exp ession o BSCL2 ansc ip s wi h exon 7 and (b)
wi hou exon 7, in WT and index case p eadipocy es, no malized o he RNA polyme ase II gene. Whi e ba : WT;
black ba : index case. All samples we e analyzed in duplica e, n = 4, *:P<0.05.
doi:10.1371/jou nal.pone.0158874.g002
Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 7/13
Senescence s udies
The pe cen age o senescen cells was calcula ed h oughou he di e en passages in o de o
assess a possible ela ionship o seipin and he c.985C>T mu a ion wi h cellula senescence.
Senescen and o al cells we e coun ed using he β-galac osidase and Hoechs 33258 dyes,
espec i ely (Fig 3). Li le SA-β-galac osidase ac i i y was de ec ed in con ol and index case
cells a P8 (P: passage, 3.9% and 3.4%, espec i ely), bu he numbe o senescen cells inc eased
con inuously and signi ican ly h ough se ial passages bo h in con ol and index case cells as
e idenced by inc eased le els o senescence-associa ed β-galac osidase ac i i y (Fig 3), which
inc eased app oxima ely 20- o 25- old, accoun ing o 76–98% o all index case cells, and 23- o
29- old, accoun ing o 78–99% o all con ol cells. This esul was expec ed as con inuous cell
di ision is known o p omo e eplica i e senescence o cells in i o [26]. No signi ican di e -
ences be ween index case and con ol p eadipocy es we e seen when cells a he same passage
we e compa ed (Fig 3).
Fig 3. Rep esen a i e pho og aphs o SA-β-gal ac i i y in p eadipocy es. Ea ly and la e passages p eadipocy es o con ol (WT) and index case
(c.985C>T) we e subjec ed o in si u SA-β-gal s aining a pH 6 and examined by b igh ield mic oscopy. The nuclei we e coun e s ained wi h Hoechs
33258. Senescen and o al cells we e quan i ied in iplica e wi h ImageJ so wa e, aking 4 andom non-o e lapping ields o iew o each sample.
O iginal magni ica ion ×10.
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Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 8/13
Exp ession o adipogenic genes a e ADSCs di e en ia ion
Di e en ia ion o ADSCs in o adipocy es o e 14 days led o accumula ion o lipid d ople s
ha we e s ained posi i ely by Oil ed O (S1 Fig). Real ime RT-PCR s udies we e used o de e -
mina e he exp ession o BSCL2 ansc ip s and adipogenic genes (Fig 4). As expec ed, ADSCs
di e en ia ion owa d he adipogenic lineage induced signi ican inc ease in he exp ession
o genes ela ed o adipogenesis: when e e ed o day 0, PPARG and LPIN1 inc eased
Fig 4. Pe cen age o change in exp ession o BSCL2 ansc ip s and adipogenic genes. Pe cen age o change in exp ession o BSCL2
ansc ip s and adipogenic genes e e ed o day 0, (a) BSCL2 wi h exon 7 (b) BSCL2 wi hou exon 7 (c) PPARG, (d) LPIN1, (e) LPL,in
ADSCs (di e en ia ed o e 14 days in o adipocy es) no malized o he 18S gene. All samples we e analyzed in duplica e, n = 4, *:P<0.05.
doi:10.1371/jou nal.pone.0158874.g004
Seipin T ansc ip s and PUFA in Celia's Encephalopa hy (PELD)
PLOS ONE | DOI:10.1371/jou nal.pone.0158874 July 8, 2016 9/13
