Immunolocalization of Substance P and NK-1 Receptor in ADIPOSE Stem Cells

 This a icle is p o ec ed by copy igh . All igh s ese ed 1 A icle IMMUNOLOCALIZATION OF SUBSTANCE P AND NK-1 RECEPTOR IN ADIPOSE STEM CELLS† Sho i le: SP/NK1R sys em in adipose s em cells Miguel Muñoz1*, Ma io F. Muñoz2 and An onio Ayala2 1 Vi gen del Rocío Uni e si y Hospi al, Resea ch Labo a o y on Neu opep ides (IBIS), Se ille, Spain 2 Depa men o Biochemis y and Molecula Biology, Uni e si y o Se ille, Spain *Co espondence: D . Miguel Muñoz Hospi al In an il Uni e si a io Vi gen del Rocío, Unidad de Cuidados In ensi os Pediá icos A . Manuel Siu o s/n 41013 - Se illa, Spain E-mail: [email p o ec ed] Phone: 34-955012965 Fax: 34-955012921 †This a icle has been accep ed o publica ion and unde gone ull pee e iew bu has no been h ough he copyedi ing, ypese ing, pagina ion and p oo eading p ocess, which may lead o di e ences be ween his e sion and he Ve sion o Reco d. Please ci e his a icle as doi: [10.1002/jcb.26134] Recei ed 20 Ma ch 2017; Re ised 12 May 2017; Accep ed 12 May 2017 Jou nal o Cellula Biochemis y This a icle is p o ec ed by copy igh . All igh s ese ed DOI 10.1002/jcb.26134  This a icle is p o ec ed by copy igh . All igh s ese ed 2 ABSTRACT Subs ance P (SP) is a neu opep ide belonging o he hachykinin pep ide amily. SP, a e binding o i s ecep o , he neu okinin 1 ecep o (NK1R), con ols se e al ansc ip ion ac o s such as NF-κB, hypoxia inducible ac o (HIF-1α), c-myc, c- os, c- jun and AP-1. SP and NK1R ha e a widesp ead dis ibu ion in bo h he cen al and pe iphe al ne ous sys ems. They a e also p esen in cells no belonging o he ne ous sys em (immune cells, placen a, e c.). SP is loca ed in all body luids, i.e., blood, ce eb ospinal luid, e c., making i ubiqui ous h oughou he human body. SP and NK1R genes a e exp essed in he s em cell line TF-1 and in p ima y s em cells de i ed om human placen al co d blood. Howe e , o ou knowledge, he p esence o SP and he NK1R ecep o in adipose s em cells (ADSC) is unknown. We demons a ed by immuno luo escence he localiza ion o SP and NK1R in human and a ADSC. SP and NK1R a e loca ed in bo h he cy oplasm and he nucleus o hese cells. The NK1R is highe in he nucleus han in he cy oplasm o ADSCs. By Wes e n blo we demons a ed he p esence o di e en iso o ms o NK1R ha ha e di e en subcellula loca ions in he ADSC. SP induces p oli e a ion and mi ogenesis h ough NK1R in ADSCs. These indings epo ed he e o he i s ime sugges an impo an ole o a SP/NK1R sys em, ei he as gene ic and/o epigene ic ac o , in bo h he cy oplasm and nucleus unc ions o he ADSCs. This a icle is p o ec ed by copy igh . All igh s ese ed Keywo ds: Subs ance P; NK1 ecep o ; adipose issue s em cells; immuno luo escence. P eamble: “Theo ies a e enewed, while he ac s emain” S. Ramon y Cajal (Nobel P ize 1906)  This a icle is p o ec ed by copy igh . All igh s ese ed 3 INTRODUCTION Subs ance P (SP) is a neu opep ide belonging o he hachykinin pep ide amily. A e binding o hei ecep o , he neu okinin-1 ecep o (NK1R), SP egula es many biological unc ions such as p oli e a ion o bo h no mal and umo cells [Munoz and Co enas, 2014] and cell mig a ion [Meshki e al., 2009]. SP and NK1R a e widely dis ibu ed in he cen al and pe iphe al ne ous sys ems, bu hey a e also p esen in o he cells and issues (immune and cance cells, placen a, and adipose issue, o example). SP is loca ed in all body luids: blood, ce eb ospinal luid, e c., making i ubiqui ous in he human body [Munoz and Co enas, 2014]. SP s imula es human pe iphe al blood monocy es o p oduce in lamma o y cy okines including in e leukin-1 (IL-1), IL-6, and umo nec osis ac o alpha (TNF-a) [Ho e al., 1996; Lau enzi e al., 1990; Lee e al., 1994; Lo z e al., 1988], which a e in ol ed in he hema opoie ic- egula ing ne wo k [Rameshwa and Gascon, 1995]. Bone ma ow (BM) cell popula ion (s oma) is pa ly esponsible o in i o hema opoie ic s imula ion by SP [Rameshwa e al., 1993]. Fib oblas s, endo helial cells, mac ophages, e icula cells, and adipocy es [Dex e e al., 1990] compose he BM s oma and p o ide he necessa y mic oen i onmen o hema opoie ic egula ion [Dex e e al., 1990]. The mechanisms in ol ed in his egula ion a e complex and appea o in ol e in e ac ions be ween he s oma and p ogeni o s/s em cells, media ed by cy okines, neu o ophic ac o s, and neu opep ide [Rameshwa and Gascon, 1995]. SP media es p oduc ion o he s em cell ac o and IL-1 in bone ma ow s oma [Rameshwa and Gascon, 1995] and has a po en s imula o y e ec on hema opoiesis [Hi amo o e al., 1998]. SP and NK1R genes a e exp essed in he human s em cell line (TF-1) and p ima y s em cells de i ed om human placen al co d blood (HPCB) [Li e al., 2000]. A e binding o NK1R, SP elici s bone ma ow s omal cell os eogenic  This a icle is p o ec ed by copy igh . All igh s ese ed 4 ac i i y, os eoclas di e en ia ion, and eso p ion ac i i y in i o in a concen a ion dependen manne [Wang e al., 2009]. I has been epo ed ecen ly ha SP enhances he p oli e a ion and mig a ion po en ial o BM mesenchymal s em cells (MSCs) [Dubon and Pa k, 2015]. Addi ionally, SP s imula es p oli e a ion o spinal neu al s em cells in spinal co d inju y ia he mi ogen-ac i a ed p o ein kinase signalling pa hway [Kim e al., 2015]. Mo eo e , SP has a p o ec i e e ec on BM MSC agains apop osis induced by se um dep i a ion [Fu e al., 2015]. Adipose issue con ains mesenchymal s em cells called adipose issue s em cells (ADSC) [Gimble e al., 2007; Lim e al., 2014; Scha le and Buchle , 2007; T ounson, 2012; T ounson and McDonald, 2015]. The p opo ion o MSC is 500 imes highe in adipose issue han in bone ma ow [F ase e al., 2006; F ase e al., 2008], so ha a la ge numbe o cells can be ob ained wi hou a la ge numbe o passes, dec easing he isk o ch omosomal abno mali ies induced senescence in cul u es [Ta e e al., 2010]. The ADSC no only ha e he po en ial o di e en ia e in o cells o mesode mal o igin and o gans, bu hey also ha e he abili y o di e en ia e in o neu ons, endoc ine cells o he panc eas, hepa ocy es, endo helial cells and ca diomyocy es [Scha le and Buchle , 2007]. ADSCs can epai and egene a e he issues by se e al mechanisms [Dimmele e al., 2014; Feiss e al., 2015; T ounson and McDonald, 2015; Tsuji e al., 2014]. The e a e no p e ious da a on whe he SP and i s ecep o a e p esen in ADSC. The e o e, he exp ession, localiza ion and dis ibu ion o NK1R and SP we e e alua ed by immuno luo escence and Wes e n blo in human and non-human ADSC. The possible e ec o SP and i s ecep o on ADSC p oli e a ion and mi ogenesis is also e alua ed.  This a icle is p o ec ed by copy igh . All igh s ese ed 5 MATERIAL AND METHODS Samples. Human adipose issue samples we e ob ained om pa ien s unde going elec i e liposuc ion su ge y and main ained a 4ºC. App o al o ob aining human issue was ob ained om a local IRB commi ee. The pa ien s signed an in o med consen o m; he esul being unanimous app o al. In he case o animals, all expe imen s we e ca ied ou acco ding o he guidelines o he Eu opean Union Council (Di ec i e 2010/63/UE) and o Spanish egula ions (BOE 34/11370, RD 53/2013) and we e app o ed by he E hic Commi ee o he Uni e si y o Se ille. Male Wis a a s (300- 500 g) we e kep a a cons an empe a u e o 22 ± 1ºC and a ela i e humidi y o 60%, wi h a ligh -da k cycle o 12 h and ee access o ood and wa e . Isola ion o S omal Vascula F ac ion (SVF). 200-300 ml o lipoaspi a e was washed wi h s e ile phospha e bu e ed saline (PBS) o emo e blood. The whole sample was cen i uged a 430 x g a 4°C o 10 min. A e cen i uga ion, he aqueous ac ion was emo ed. The lipid ac ions we e dilu ed wi h an equal olume o collagenase solu ion (Collagenase NB 4G, Se a) 0.3 U/ml in PBS (Wünsch uni s). The mix u e was incuba ed a 37°C o 60 min in an o bi al shake . A e diges ion, an equal olume o Dulbecco's Modi ied Eagles Medium (DMEM) con aining 20% e al bo ine se um (FBS) was added. The mix u e was cen i uged a 600 x g o 10 min and he pelle was esuspended in 10 ml o expansion medium o non-hema opoie ic s em cells (NH- Mil enyi Bio ec). Cell suspension was passed h ough a 100 µm il e (BD Biosciences). The il a e was cen i uged a 300 x g o 10 min. The pelle was esuspended in 5 ml o NH medium and passed h ough a 40 µm il e . Finally, cell numbe s we e de e mined in his cell suspension. Magne ic sepa a ion o Human ADSC (hADSC). ADSC we e magne ically labeled by using CD271 (LNGFR)-APC an ibodies and An i-APC Mic oBeads (Mil enyi  This a icle is p o ec ed by copy igh . All igh s ese ed 6 Bio ec). CD271 is a well-known ma ke on mesenchymal s em cells, also known as mesenchymal s omal cells (MSCs), om bone ma ow aspi a e o lipoaspi a e [Qui ici e al., 2002]. The sepa a ion is pe o med in columns inse ed in an ex emely s ong magne , which induces in he column ma ix a su icien ly high in ense magne ic ield o e aining he labeled cells. Unlabeled cells pass eely h ough he column and a e disca ded. A e emo ing he column om he magne , cells a e elu ed wi h washing bu e . The p ocedu e is as ollows: The cell suspension was cen i uged a 300 x g o 10 min. The pelle was esuspended in 80 μl o inse bu e (Mil enyi Bio ec). Ten μl o CD271-APC an ibody we e added. A e 10 min a 4ºC, cells we e cen i uged a 300 x g o 10 min. Cells a e esuspended wi h 70 μl o bu e inse and 20 μl o An i-APC Mic obeads we e added. The samples we e incuba ed o 15 min a 4°C. Cells we e washed and cen i uged a 300 x g o 10 min. Finally, he pelle was esuspended in 500 μl o wash bu e and he sample was passed h ough he column inse ed in he magne ic ield acco ding o he manu ac u e ´s ins uc ions. Cell expansion. A e magne ic sepa a ion, cells we e cen i uged a 350 x g and esuspended in 1 ml o NH expansion medium p ehea ed a 37°C. The cells we e cul u ed in a 25 cm2 lask by adding 4 ml o NH Expansion Medium. Passaging o hADSC. Cell cul u es we e washed wice wi h PBS. Enough olume o T ypsin / EDTA (0.05% / 0.53 mM) (Gibco) was added o co e he cells and hey we e hen incuba ed a 37°C o 5 min. A e wa ds, DMEM wi h 20% FBS was added and he sample was cen i uged a 300 x g o 10 min a oom empe a u e. The cells we e esuspended in 2 ml o NH expansion medium. Ra S omal Vascula F ac ion. Animals we e anaes he ized wi h ke amine/xylazine (100/12.5 mg/kg). Adipose issue o he abdominal o lumba egion was ob ained and  This a icle is p o ec ed by copy igh . All igh s ese ed 7 immedia ely washed wi h 20 ml o PBS o emo e blood. Fa was ea ed as human samples o ob ain SVF. Labeling and magne ic sepa a ion o a ADSC ( ADSC). Magne ic isola ion o ADSC was ca ied ou by he same p ocedu e used o hADSC. In his case, he an ibody was agains he su ace an igen CD90.1 ( hymocy e di e en ia ion an igen- 1.1) o ADSC cells. The mouse monoclonal an ibody eac s wi h a CD90 (Thy-1), a GPI-ancho ed conse ed glycop o ein memb ane. In he a , he CD90 an igen is exp essed on hymocy es, ecen hymic emig an s, hema opoie ic s em cells, neu ons and mesenchymal s omal cells [Da ies e al., 2015]. Cell Expansion. The p o ocol o cell expansion was he same as o human, using DMEM, 20% FBS and 1% an ibio ic as cul u e medium. Pheno yping o hADSC. Pheno yping o ADSC was ca ied ou by low cy ome y and a comme cial ki (MSC Pheno yping Ki human, Mil enyiBio ec) con aining he necessa y an ibodies and luo opho es. The luo opho e-conjuga ed monoclonal an ibodies we e: CD14-Pe CP, CD20-Pe CP, CD34-Pe CP, CD45-Pe CP, CD73-APC, CD90-FITC and CD105-PE. Pheno yping o ADSC. In his case he cells mus be posi i e o CD90.1 and CD29 and nega i e o CD11b, CD34, and CD45. The monoclonal an ibodies we e he ollowing: an i-CD34-PE (San aC uz, USA), an i-CD45-PE (BD Pha mingen), an i- CD11b-PE (Bio-Legend), an i-CD29-APC (Mil enyi Bio ec) and an i-CD90.1-FITC (Mil enyi Bio ec). Assay o hADSC di e en ia ion in o adipocy es. The cells we e dilu ed wi h adipocy es di e en ia ion medium (Mil enyi Bio ec). The medium was changed e e y h ee days. On he 21s day, adipogenic di e en ia ion was con i med by he o ma ion o neu al lipid- acuoles s ainable wi h Oil Red O (Sigma Ald ich, USA).  This a icle is p o ec ed by copy igh . All igh s ese ed 8 Assay o hADSC di e en ia ion in o Os eoblas . Cells we e dilu ed wi h os eoblas di e en ia ion medium (Mil enyi Bio ec). On he 10 hday, os eogenesis was assessed by phospha ase alkaline s aining (Sigma Ald ich). Di e en ia ion assays in ADSC. Fo di e en ia ion in o adipocy es, cells we e cul u ed in di e en ia ion medium (S emXVi o Media-R&D Sys em) wi h added adipogenic supplemen s con ained in he ki . De ec ion was pe o med as indica ed o hADSC. Fo os eaoblas di e en ia ion assay, cells we e cul u ed in he same medium plus os eogenic supplemen s. Phospha ase alkaline de ec ion was he same as hADSC. Immuno luo escence de e mina ion in cul u e cell. Cells we e cul u ed in a 24-well pla e con aining gela in-coa ed co e slips. When cells eached 80% con luence, he cul u e media we e emo ed om each well and washed wice wi h PBS. One ml o ixa i e solu ion (4% pa a o maldehyde) was added o each well and he pla es we e kep o 20 min a 4°C. Wells we e washed wice wi h PBS and incuba ed wi h blocking solu ion (PBS plus 1% e al bo ine se um) o 1 h a oom empe a u e. A e emo ing he blocking solu ion, each well was washed 3 imes wi h PBS. Cells we e incuba ed wi h abbi -de i ed an i-NK1R (which speci ically co esponds o esidues KTMTESSSFYSNMLA, co esponding o he C- e minus o NK1R (amino acids 393- 407)) (Sigma, 1:1000) o abbi -de i ed an i-Subs ance P (Millipo e, 1:1000), he p esence o 10% se um and 0.25% T i on X-100 a 4°C o e nigh . The wells we e washed 3 imes wi h PBS and incuba ed wi h he an i- abbi seconda y an ibody conjuga ed o Alexa Fluo 488 (The mo Fishe ) dilu ed 1:500 in 0.25% T i on X-100 and Hoechs (Sigma) dilu ed 1: 2000 in PBS o 1 h a 37°C. A e incuba ion, he samples we e washed 3 imes wi h PBS and moun ed wi h moun ing luid o luo escence (DAKO). Samples we e isualized in a con ocal mic oscope (Zeiss LSM7  This a icle is p o ec ed by copy igh . All igh s ese ed 9 DUO). As nega i e con ol, he p ima y an ibody was omi ed and eplaced by non- immune se um. Subcellula ac iona ion. Cells we e de ached by sc apping in cold PBS and we e cen i uged a 15,000g o 20 s a 4ºC and esuspended in bu e A (10 mM Hepes, 2 mM MgCl2, 15 mM KCl, 0.1 mM EDTA, 0.1% NP-40, 250 mM Suc ose, 1 mM DTT; pH 7.6) con aining p o ease inhibi o s and incuba ed 7 min on ice. Cells we e cen i uged a 1,000 x g o 10 min o sepa a e he cy oplasmic ac ion o he cell ex ac . The nuclei we e lysed by incuba ion on ice o 45 min wi h RIPA bu e (20 mM T is–HCl, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxychola e, 2.5 mM sodium py ophospha e, and 1 mM sodium o ho anada e) con aining p o ease inhibi o s. The lysa e was hen cen i uged a 15,000 x g o 20 min. The esul ing supe na an was used as he nuclea ac ion o he cell ex ac . The subcellula ac ions we e sepa a ed by SDS–PAGE and analyzed by immunoblo using an ibodies agains cy oplasmic and nuclea ma ke p o eins. Immunoblo . P o ein con en o he samples was es ima ed wi h a Pie ce BCA p o ein assay ki (Pie ce Bio echnology, USA). P o ein samples we e sepa a ed by SDS–PAGE (12% ac ylamide) and ans e ed o a PVDF memb ane (GE Heal hca e, USA) a 100 V o 1 h. The memb anes we e incuba ed wi h blocking bu e (5% d y milk in 20 mM T is–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20) o 1 h a oom empe a u e. Memb anes we e hen incuba ed o e nigh a 4ºC in blocking solu ion con aining he ollowing an ibodies: NK1R (1:2000), α- ubulin (1:3000), β-ac in (1:3000; Sigma), and hnRNP (1:2000; San a C uz Bio echnology, San a C uz, CA, USA). A e incuba ion, he memb anes we e washed in 20 mM T is–HCl, pH 7.5, 500 mM NaCl, 0.05% Tween 20 and incuba ed wi h pe oxidase-conjuga ed an i-  This a icle is p o ec ed by copy igh . All igh s ese ed 16 impo an in cell sp eading and cell mig a ion [Meshki e al., 2009]. Al hough he abo e men ioned mechanisms can explain he immunolocaliza ion o SP and NK1R in he cy oplasm o s em cells, he meaning o he loca ion o SP and NK1R inside s em cell nuclei emains unknown. Because SP and NK1R egula e se e al cy oplasmic signalling pa hways ex acellula ly, he p esence o SP and NK1R inside he s em cell nucleus is heo e ically unnecessa y. I is known ha localiza ion o a pep ide and i s ecep o is in ol ed in he unc ion o he cellula compa men whe e hey a e loca ed. Thus, he p esence o SP and i s ecep o in he nucleus o s em cells sugges s ha bo h a e in ol ed in he nuclea unc ion. Ou esul s also show a posi i e co ela ion be ween DNA concen a ion and NK1R exp ession (Fig. 8D), sugges ing he possible in ol emen o nuclea NK1R in he mi ogenesis p ocess. In addi ion, i is known ha SP exe s i s e ec only once i is bound o i s ecep o and bo h a e ound in he nucleus o s em cells. This means ha he nucleus o ADSC con ains e e y hing needed o he SP o wo k and hus SP migh egula e gene exp ession o s em cells. Simila ly o ha , SP modula es emo ional conduc unde con ol o he limbic sys em, he p esence o SP in he cell nucleus could be in ol ed in s em cell beha io . Up o now, he neu o ansmi e , neu opep ides and neu omodula o s we e exclusi ely conside ed as cell signaling molecules in mul icellula o ganisms. Howe e , hese indings demons a e ha neu opep ides a e no exclusi e o mul icellula o ganisms. Indeed, he localiza ion o SP and NK1R in s em cell nuclei opens he doo o unde s anding ha s em cells possibly ha e a udimen a y b ain cell. Fu he mo e, i has been epo ed ha SP, calci onin gene ela ed pep ide (CGRP) and NK1R a e loca ed in he nucleus o he do sal oo ganglia subpopula ion cells in a s. Howe e , he physiological signi icance emains unknown [Boe and  This a icle is p o ec ed by copy igh . All igh s ese ed 17 Gon ijo, 2006]. Also, i has been epo ed ha SP and NK1R a e loca ed in bo h no mal and umo cell nuclei [B ene e al., 2009; Es eban e al., 2009; Munoz e al., 2013; Munoz and Co enas, 2014; Munoz e al., 2012; Munoz e al., 2014; Munoz e al., 2010]. Due o i s nuclea localiza ion, SP has been conside ed a gene ic neu omodula o [Munoz e al., 2010]. He e, we ha e demons a ed ha nuclea localiza ion o SP and NK1R is highe han in cy oplasm o adipose s em cells. This sugges s ha SP has bo h cy oplasmic and nuclea unc ions; mos likely he nuclea unc ion is he mo e impo an since he SP e ec is concen a ion-dependen . Addi ionally, i has been epo ed ha SP egula es some ansc ip ion ac o s such as NF-κB, c-myc, c- os, c-jun, AP-1, and hypoxia- inducible ac o (HIF-1α) ha a e in ol ed in in lamma ion (NF-kB), mi ogenesis and cellula di e en ia ion (c- os, c- jun and AP-1) and angiogenesis and asculogenesis (HIF-1α) [Koh e al., 2010; Luo e al., 1996; Munoz e al., 2015; Walczak-D zewiecka e al., 2008]. The exp ession o SP in he nuclei o adipose s em cells sugges s ha SP could play wo oles in modi ying he gene exp ession o adipose s em cells: indi ec ly, by egula ing ansc ip ion ac o s and di ec ly, ia nuclea NK1R egula ion. These indings a e in ag eemen wi h he high p esence o SP and NK1R wi hin he nucleus o no mal and umou cells [Munoz e al., 2015]. This means ha he SP pep ide h ough he NK1R could egula e he nuclea ac i i y o s em cells and ha SP could ac ei he as gene ic o epigene ic nuclea ac o o egula e gene exp ession. In ega ds o i s epigene ic ole, i has been desc ibed ha SP induces he exp ession o some gene ia his one deace ylase ac i i y in colonic epi helial cells [Koon e al., 2007]. Indeed, he inc eased exp ession o NK1R in he nucleus o ADSC du ing mi osis o e he le els ound in non-di iding cells (Fig. 6) demons a ed ha he neu ogenic ac o s (SP) could egula e he gene ic cell cycle machine y o s em cells.  This a icle is p o ec ed by copy igh . All igh s ese ed 18 In summa y, we demons a e he e o he i s ime he immunolocaliza ion o SP and NK1R in human and a ADSCs. Bo h a e loca ed in he cy oplasm and he nucleus o hese cells. The immunolocaliza ion o he NK1R is highe in he nucleus han in he cy oplasm o ADSCs. The iso o m o 58 kDa NK1R iso o m was mainly p esen in he nucleus, whe eas he le el o he 33 kDa iso o m was highe in he cy oplasm. SP induces p oli e a ion and mi ogenesis h ough NK1R in ADSCs. Taken oge he , hese indings could sugges a ole o SP/NK1R sys ems in he cy oplasm and nucleus unc ions o he ADSCs such as p oli e a ion, di e en ia ion and mig a ion. ACKNOWLEDGEMENTS This wo k was suppo ed by P oyec o de Excelencia de la Jun a de Andalucia (P10- CTS-6494).  This a icle is p o ec ed by copy igh . All igh s ese ed 19 REFERENCES Boe PA, Gon ijo JA. 2006. Nuclea localiza ion o SP, CGRP, and NK1R in a subpopula ion o do sal oo ganglia subpopula ion cells in a s. Cell Mol Neu obiol 26:191-207. 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Os eoblas di e en ia ion is de ec ed by Alkaline Phospha ase s aining (C, G). Flow Cy ome y analysis show ha almos all hADSCcul u es exp ess CD73/CD90 (99.6%) and CD90/CD105 (99.7%), while a small po ion o hADSC exp ess nega i e ma ke s (0.11%) (D). ADSC exp ess CD29/CD90.1 (99.1%) and nega i e ma ke s (10.3%) (H). Scale ba = 50 µm. Figu e 2. Immuno luo escence localiza ion o SP in ADSCs. Localiza ion o SP(g een) in hADSC and ADSC. Nuclei (Blue) we e s ained by Hoeschs . Scale ba = 50 µm. Figu e 3. Immuno luo escence localiza ion o NK1R in ADSCs. Localiza ion o NK- 1R (g een) in hADSC and ADSC. Nuclei (Blue) a e s ained by Hoeschs . Scale ba = 50 µm. Figu e 4. In ensi y p o iles o SP in ADSCs. Immuno luo escence in ensi y p o ile in hADSC and ADSC o SP (g een) was measu ed using Zen Applica ion 2011 so wa e. The lines we e placed ac oss cells and he simul aneously occu ing in ensi y peaks we e e alua ed. Line scan g aphs show he immuno luo escence in ensi y along he posi ioned whi e a ow a e sub ac ion o he backg ound. Scale ba = 50 µm. Figu e 5. In ensi y p o iles o NK1R in ADSCs. Immuno luo escence in ensi y p o ile in hADSC and ADSCs o NK-1R (g een) was measu ed using Zen Applica ion 2011 so wa e. The lines we e placed ac oss cells and he simul aneously occu ing in ensi y  This a icle is p o ec ed by copy igh . All igh s ese ed 23 peaks we e e alua ed. Line scan g aphs show he immuno luo escence in ensi y along he posi ioned whi e a ow a e sub ac ion o he backg ound. Scale ba = 50 µm. Figu e 6. In ensi y p o iles o NK1R in ADSCs du ing he p ocess o mi osis. Immuno luo escence in ensi y p o ile in ADSC and o NK-1R (g een) was measu ed using Zen Applica ion 2011 so wa e. Line scan g aphs show he immuno luo escence in ensi y along he posi ioned whi e a ow. Scale ba = 50 µm. Figu e 7. E idence o subcellula localiza ion o NK1R by WB. a-Tub (50 kDa) and hnRNP (37 kDa) we e used as cy oplasmic and nuclea ma ke s while β-Ac in (40 kDa) was used as loading con ol. In addi ion, wo bands o NK1R we e obse ed wi h di e en molecula weigh (33 kDa and 58 kDa). The op ical densi ies o NK1R bands we e di ided by he op ical densi ies o β-ac in. The esul s a e he mean ± SEM o h ee independen subcellula loca ion expe imen s. W: Whole cell ac ion, C: Cy oplasmic ac ion and N: Nuclea ac ion. a: s a is ically di e en om whole cells. b: s a is ically di e en om cy oplasm. Figu e 8. P oli e a i e e ec o SP and mi ogenic in ol emen o NKR1 in ADSCs. MTS iabili y assay was pe o med in oc uplica e echnical eplica es pe g oup in h ee independen expe imen s. Resul s a e exp essed as pe cen age wi h espec o he amoun ound in con ol (N=3, *p=0.001) (A). ADSCs popula ion labeled wi h NK1R- FITC was ga ed as R1 in FSC agains SSC do plo (double s disc imina ion was emo ed using an AUX channel). The R1 popula ion was ep esen ed in his og ams o PI lin and NK1R FITC Log (g ey colo ep esen s he luo escence in ensi y o seconda y an ibody con ol) (B). PI lin his og am was ga ed in o egions R2 (less PI in ensi y) and R3 (mo e PI in ensi y) and bo h we e ga ed in NK1R FITC Log his og ams (C). Mean luo escence in ensi y o NK1R-FITC was ob ained om h ee independen expe imen s (N=3, *p<0.001) (D).  This a icle is p o ec ed by copy igh . All igh s ese ed 24 Figu e 1  This a icle is p o ec ed by copy igh . All igh s ese ed 25 Figu e 2