ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube For Chemical Industry chemical component, Kuperewera kwa SPECC1L kumabweretsa kukhazikika kwamagulu ophatikizika ndikuchepetsa kukhetsedwa kwa ma cell a cranial neural crest.

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ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded chube Kwa Makampani Amankhwala

Liaocheng Sihe SS Material Co., Ltd.ndi opanga otsogola omwe ali otsogola mu mapaipi osapanga zitsulo zosapanga dzimbiri, machubu owala, machubu opanda msokonezo ndi zina.Kuti tithandizire makasitomala, tili ndi mipope yowotcherera komanso machubu.Liaocheng Sihe SS Material Co., Ltd.ali ndi zida zapamwamba kwambiri zopangira ndi kuyesa.Titha kukwaniritsa zomwe mukufuna.Malinga ndi muyezo mosamalitsa, machubu omwe timapanga nthawi zonse amakhala ndi kulolerana kolondola kwa OD ndi WT.The kulolerana ulamuliro mosamalitsa malinga kubala miyezo.Zogulitsa zathu nthawi zonse zimakhutitsidwa ndi makasitomala.Makasitomala adagula zinthu zathu adapanga phindu lochulukirapo.
a) OD (Kunja Diameter): 3.18mm mpaka 101.6mm
b) WT (Wall Makulidwe): 0.5mm mpaka 20mm
c) Utali: Malinga ndi zomwe kasitomala amafuna
d) Miyezo : ASTM A312;ASTM A269;ASTM A789;ASTM A790 ndi zina
e) Njira Yopangira: ERW, EFW etc

Masilayidi owonetsa zolemba zitatu pa slide iliyonse.Gwiritsani ntchito mabatani akumbuyo ndi ena kuti mudutse zithunzi, kapena mabatani owongolera masilayidi kumapeto kuti mudutse silayidi iliyonse.
Ma cell a cranial neural crest cell (CNCC) amatsika pang'onopang'ono kuchokera ku ma embryonic neural folds ndikusamukira ku pharyngeal arches, omwe amapanga mbali zambiri zapakati.Kukanika kwa CNCC kumatenga gawo lofunikira mu etiology ya orofacial cleft, malformation wamba wobadwa nawo.Kusintha kwa Heterozygous SPECC1L kwapezeka mwa odwala omwe ali ndi ma atypical and syndromic clefts.Apa, tikuwonetsa kukhathamiritsa kwazinthu zomatira zomatira (AJ), β-catenin ndi E-cadherin m'maselo ogogoda a SPECC1L, ndi ma electron micrographs amawonetsa kufalikira kwa apical-basal kwa AJ.Kuti timvetsetse udindo wa SPECC1L mu craniofacial morphogenesis, tidapanga mtundu wa mbewa wopanda Specc1l.Homozygous mutants ndi owopsa ndipo amawonetsa kutsekedwa kwa neural chubu ndi CNCC lamination.Kudetsa kwa mapuloteni a AJ kumachulukitsidwa mu mutant neural folds.Chilema cha AJ ichi chimagwirizana ndi cholakwika cha CNCC delamination, chomwe chimafuna kuyimitsidwa kwa AJ.Kuphatikiza apo, zosintha za Specc11 zachepetsa kusaina kwa PI3K-AKT ndikuwonjezera apoptosis.Mu vitro, kuletsa pang'ono kwa PI3K-AKT kusaina m'maselo akutchire kunali kokwanira kupangitsa kusintha kwa AJ.Chofunika kwambiri, kusintha kwa AJ koyambitsidwa ndi kugwetsa kwa SPECC1L kumatha kusinthidwa ndikutsegula njira ya PI3K-AKT.Kuphatikizidwa pamodzi, izi zikuwonetsa kuti SPECC1L, monga chowongolera chaposachedwa cha PI3K-AKT siginecha ndi AJ biology, ikufunika kuti neural chubu kutsekedwa ndi CNCC stratification.
Maselo a Cranial neural crest cell (CNCCs) amapezeka ku dorsal neuroectoderm ndikuchotsa ku neuroepithelium ya neural folds yomwe ikukula kudzera munjira yomwe imakhudza kusintha kwa epithelial-mesenchymal (EMT)1,2,3.Ma CNCC otsogolera a epithelial amasokoneza ma intercellular intercellular ndikukhala mesenchymal CNCCs omwe amadzaza mitsempha yoyamba ndi yachiwiri ya pharyngeal ndi kupanga kwambiri craniofacial cartilage.Chifukwa chake, majini omwe amawongolera ntchito ya CNCC nthawi zambiri amasokonekera mu etiology ya craniofacial congenital anomalies monga orofacial clefts, zomwe zimakhudza obadwa 1/800 ku US kokha.Chimodzi mwa zolakwika zobadwa nazo8.
Delamination ya CNCC ikugwirizana ndi kutsekedwa kwa anterior neural chubu pakati pa 8.5 ndi 9.5 masiku a chitukuko cha embryonic mu mbewa.Zosinthika zamitundu ingapo yolumikizana ndi mbewa za orofacial cleft zimawonetsanso mtundu wina wa neural tube defect, kuphatikiza Irf69,10, Ghrl310, Cfl111, ndi Pdgfrα12.Komabe, njira zotsekera neural chubu ndi CNCC stratification zitha kuonedwa ngati zodziyimira pawokha, monga mbewa ya Splotch mutant (Pax3) ikuwonetsa zolakwika mu kutsekedwa kwa neural chubu popanda zotsatira za CNCC stratification kapena kusamuka 13,14.Mitundu yowonjezera ya mbewa yokhala ndi zolakwika mu CNCC dissection ndi kutsekedwa kwa neural chubu zithandizira kufotokozera momwe maselo amakhalira panjira ziwirizi.
Kupatulidwa kwa CNCC ku maselo a neuroepithelial kumafuna kusungunuka kwa zomatira (AJs), zomwe zimakhala ndi mapuloteni opangidwa ndi mapuloteni omwe ali, mwa ena, E-cadherin, β-catenin, α-E-catenin, ndi α-actinin yogwirizana ndi actin filaments 2. Maphunziro owonjezera a E-cadherin m'makutu a neural amasonyeza kuchepetsa kapena kuchedwa kwa CNCC delamination.Mosiyana ndi zimenezi, kuponderezedwa kwa E-cadherin kumabweretsa kusamvana koyambirira15,16.Zambiri zomwe zimayimira EMT panthawi ya CNCC stratification ndi zinthu zolembera (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) ndi mapuloteni okonzanso a extracellular matrix (ECM) monga matrix metalloproteinases (MMPs), komabe CNCCs ndi cytoskeletal AJ regulators. sichinadziwikebe.Njira ya PI3K-AKT imadziwika kuti imasokoneza milingo ya E-cadherin, makamaka kuchokera ku kafukufuku wa khansa17.Kafukufuku waposachedwapa wasonyeza kuti kutayika kwa PDGFα-based PI3K-AKT signing mu mbewa kumabweretsa zovuta za craniofacial, kuphatikizapo cleft palate ndi neural tube defects12.Komabe, ubale pakati pa njira ya PI3K-AKT ndi kukhazikika kwa AJ pa CNCC stratification sizikudziwika.
M'mbuyomu tidazindikira SPECC1L ngati jini yoyamba yosinthika mwa anthu awiri okhala ndi mng'alu waukulu womwe umachokera pakamwa mpaka diso, lotchedwa oblique cleft (ObFC) kapena Tessier IV18 cleft.Kusintha kwa SPECC1L kwadziwika m'mabanja awiri amitundu yosiyanasiyana omwe ali ndi autosomal dominant Opitz G/BBB syndrome (OMIM #145410), momwe anthu okhudzidwa amawonetsa hyperdistance ndi cleft lip/palate19, komanso m'banja limodzi lomwe lili ndi matenda a Tibi overdistance (OMIM #145420)20 .oposa theka la matenda a Opitz G/BBB ndi X-linked (OMIM #300000) ndipo amayamba chifukwa cha masinthidwe amtundu wa MID1, womwe umayika mapuloteni 22 a cell skeleton yogwirizana ndi microtubule.Timalingalira kuti SPECC1L, yomwenso ndi mapuloteni okhudzana ndi ma microtubules ndi actin cytoskeleton, akhoza kugwirizanitsa ma signature ofunikira kuti actin cytoskeleton ikonzedwenso panthawi yolumikizana ndi selo ndi kusamuka 18.Kupyolera mu maphunziro a in vitro ndi mu vivo, tsopano tikufotokoza SPECC1L ngati chowongolera chatsopano cha kukhazikika kwa AJ kudzera pa siginecha ya PI3K-AKT.Pa mlingo wa ma cell, kuchepa kwa SPECC1L kunapangitsa kuchepa kwa mapuloteni a pan-AKT ndi kuwonjezeka kwa apical-basal dispersion ya AJ, yomwe inathetsedwa ndi kuyambitsa kwa mankhwala kwa njira ya AKT.Mu vivo, mazira omwe alibe Specc11 amawonetsa kutsekedwa kwa neural chubu ndikuchepetsa kugawanika kwa CNCC.Chifukwa chake, SPECC1L imagwira ntchito pamasanjidwe okhazikika a cell omwe amafunikira kuti azigwira bwino ntchito pa CNCC pa nkhope ya morphogenesis.
Kuti tiwonetsere gawo la SPECC1L pama cell cell, tidagwiritsa ntchito mzere wokhazikika wa osteosarcoma cell U2OS woperewera mu SPECC1L18.Maselo okhazikika a U2OS omwe ali ndi SPECC1L (kd) kugogoda anali ndi kuchepa kwapang'onopang'ono (60-70%) m'magulu a SPECC1L zolemba ndi mapuloteni, pamodzi ndi zolakwika za kusamuka ndi kukonzanso kwa actin cytoskeleton 18. Mosiyana, kuchepa kwakukulu kwapang'onopang'ono SPECC1L yawonetsedwa kuti imatsogolera ku zolakwika za mitotic 23.Titadziwikanso, tidapeza kuti maselo athu okhazikika a SPECC1L-kd adasintha morphology pamlingo wapamwamba kwambiri (Chithunzi 1).Maselo owongolera payekha ndi ma cell a kd pakulumikizana kochepa amawoneka ofanana (Chithunzi 1A, D).Maola 24 pambuyo maphatikizidwe, kulamulira maselo anapitiriza mawonekedwe awo cuboidal (mkuyu. 1B, E), pamene SPECC1L-kd maselo elongated (mkuyu. 1C, F).Kukula kwakusintha kwa mawonekedwe a cell kudatengedwa ndi zithunzi zamoyo zama cell ndi ma cell a kd (kanema 1).Kuti tidziwe udindo wa SPECC1L m'maselo ophatikizika, tidawunika kaye kafotokozedwe kake.Tidapeza kuti mapuloteni a SPECC1L adakula pakuphatikiza (Chithunzi 1G), pomwe milingo ya SPECC1L sinachuluke (Chithunzi 1H).Kuonjezera apo, pamene kuchuluka kwa maselo kumawonjezeka, mapuloteni a SPECC1L amasonkhanitsidwa pamalire a intercellular (mkuyu 2A-E), ndi ndondomeko yomwe ikugwirizana ndi membrane-yogwirizana ndi β-catenin (Mkuyu 2A'-E ').Poganizira kuyanjana kwa SPECC1L ndi actin cytoskeleton 18,23 tinaganiza kuti SPECC1L imalumikizana ndi ma actin-based adhesive junctions (AJ).
(AF) SPECC1L knockdown (DF) maselo amatalika pa confluence kwambiri (F) poyerekeza ndi kulamulira U2OS maselo (AC).Zomwe zikuwonetsedwa apa ndi mfundo zitatu mwa zisanu ndi chimodzi (T1, T3, T6) zomwe tidasankha pama cell osiyanasiyana.(G) Kusanthula kwa kumadzulo kwa Western blot kusonyeza kuti mapuloteni a SPECC1L amakhazikika pamlingo wapamwamba wa confluence poyerekeza ndi chiwerengero chochepa cha kugwirizana kwa maselo olamulira.Western blot ya SPECC1L ikuwonetsa bandi yoyembekezeka ya 120 kDa ndi bandi yolemera kwambiri ya mamolekyulu, mwina atasinthidwa pambuyo pomasulira (*).Kusanthula kwa Western blot kunachitika pansi pamikhalidwe yomweyi pakulumikizana kochepa komanso kwakukulu.Zithunzi zosonyeza SPECC1L pakulumikizana kotsika komanso kwapamwamba zidatengedwa kuchokera pachitsamba chomwecho.Chotupa chomwechi chinachotsedwa ndikuwunikidwanso ndi β-actin antibody.(H) Kusanthula kochuluka kwa RT-PCR sikunawonetse kusintha kwakukulu pamiyeso ya SPECC1L yolembera.Mipiringidzo yolakwika imayimira ma SEM kuchokera pazoyeserera zinayi zodziyimira pawokha.
(AE) Tidasankha nthawi zisanu ndi imodzi (T1-T6) zomwe zikuyimira kuchuluka kwa ma cell kuti asinthe mawonekedwe a cell ndikusintha kwa AJ m'maselo a U2OS okhala ndi SPECC1L knockdown (kd).Zoyamba zisanu za nthawiyi zinaphatikizapo maselo amodzi (T1), 50-70% kuphatikizika kwamagulu ang'onoang'ono a maselo (T2), kusakanikirana popanda kukonzanso maselo a kd (T3), kukonzanso maselo a kd (T4), ndi kusintha kwa maola 24.mu mawonekedwe apambuyo a kd (T5) maselo.Puloteni ya SPECC1L idamwazikana makamaka mu cytoplasm ku T1 (A), koma kudzikundikira kwake kunkawonedwa pamalire a intercellular pamagawo otsatirawa (B-E, mivi).(FJ) β-catenin imasonyeza kudzikundikira kofanana pamalire a intercellular okhudzana ndi zovuta za AJ.(A'-E') SPECC1L ndi β-catenin amawonetsa madontho opitilira malire pama cell pama cell kwambiri (mivi).(F'-J') M'maselo a SPECC1L-kd, kuyatsa kwa β-catenin kumawoneka ngati kwabwinoko pakachulukidwe ka cell (F'-H'), koma kumakulirakulira pamene mawonekedwe a cell akusintha (I', J'; mivi), kusonyeza kuti AJ zasintha.Mipiringidzo = 10 µm.
Kenako tinayesa kudziwa zotsatira za kuchepa kwa SPECC1L pa AJ.Tinagwiritsa ntchito zizindikiro zingapo zogwirizana ndi AJ, kuphatikizapo zigawo za canonical F-actin, myosin IIb, β-catenin, ndi E-cadherin24,25,26,27.Mitsempha ya actin stress inawonjezeka m'maselo a SPECC1L-kd monga tafotokozera kale (Mkuyu 3A, B) 18.Myosin IIb yokhudzana ndi actin filaments inasonyeza kuwonjezeka kofanana kwa maselo a SPECC1L-kd mu vitro (Mkuyu 3C, D).β-catenin yogwirizana ndi AJ imamangiriza ku cadherin pa nembanemba ya selo, kusonyeza "chisa cha uchi" chowonetseratu mu control cubocytes (Mkuyu 3E, G).Chochititsa chidwi n'chakuti, pazithunzi zojambulidwa pogwiritsa ntchito microscopy confocal, β-catenin (Fig. 3E, F) ndi E-cadherin (Fig. 3G, H) yodetsa pa cell membrane ya confluent SPECC1L-osowa maselo akuwonetsa machitidwe odziwika a madontho otalikirapo.Kukula kwa AJ-ogwirizana ndi β-catenin kutayira m'maselo a kd kunkadziwika kwambiri pa confluence, koma kumawoneka kuti kutsogolere kusintha kwa mawonekedwe a selo (Mkuyu 2F-J, F'-J').Kuti tidziwe mawonekedwe amtundu wa AJ wotalikirapo, tidawunika malire a cell pa apical-basal pamwamba pa ma cell a SPECC1L-kd U2OS pogwiritsa ntchito ma electron microscopy (TEM) (Chithunzi 3I, J).Mosiyana ndi kulamulira maselo (mkuyu. 3I), amene anali osiyana elekitironi wandiweyani zigawo zosonyeza AJ (mivi), kd maselo (mkuyu. 3J) anasonyeza lalikulu, contiguous zigawo za mkulu ma elekitironi kachulukidwe chosonyeza AJ pamodzi apicobasal ndege..Kuonjezera apo, pazigawo zodutsamo, tinawona mapepala ochuluka a cell membrane m'maselo a kd (mkuyu S1A, B), yomwe imalongosola ndondomeko yowonjezera ya β-catenin ndi E-cadherin staining bands (Mkuyu 3F, H).Pothandizira udindo wa SPECC1L mu AJs, β-catenin anali co-immunoprecipitated ndi SPECC1L mu lysates ya confluent U2OS maselo (Fig. 3K).Pamodzi ndi kuwonjezereka kwa chitetezo chamthupi kwa zolembera za AJ, kusanthula kwa TEM kunali kogwirizana ndi lingaliro lathu lakuti kuchepa kwa SPECC1L kumawonjezera AJ apical-basal density ndi kusiyana.
(AH) Kuchulukitsa madontho a F-actin m'maselo a kd pa maola 48 atatha kuphatikizika (T6; A, B).Madontho osinthika a myosin IIb okhudzana ndi F-actin (C, D).Mawonekedwe osalala a β-catenin ndi E-cadherin membrane amadetsa m'maselo owongolera (E, G) adalimbikitsidwa m'maselo a SPECC1L-kd (F, H).Mipiringidzo = 10 µm.(I-J) Ma electron micrographs omwe amayang'ana mbali ya apical-basal intercellular.Maselo owongolera amawonetsa zigawo zowirira kwambiri za ma elekitironi zomwe zikuwonetsa zomata (I, mivi).Mosiyana ndi izi, mphambano yonse ya apical-basal mu SPECC1L-kd ma cell adawoneka ngati electron wandiweyani (J, mivi), kusonyeza kuchulukirachulukira komanso kubalalitsidwa kwa zomatira.(K) β-catenin anali co-immunoprecipitated ndi SPECC1L mu confluent U2OS cell lysates.Chithunzi chojambulidwa kuchokera pamalo amodzi choyimira chimodzi mwa zoyeserera zinayi zodziyimira pawokha.
Kuti timvetse udindo wa SPECC1L mu craniofacial morphogenesis, tinapanga chitsanzo cha mbewa chosowa cha Specc1l pogwiritsa ntchito mizere iwiri yodziimira ya ES trap cell, DTM096 ndi RRH048 (BayGenomics, CA), yomwe imayimira intron 1 ndi Specc1l zolemba zinagwidwa pa 15 (Mkuyu 1) .4A, chithunzi S2).Ma genomic malo a decoy vector insert adatsimikiziridwa ndi kutsatizana kwa ma genome ndikutsimikiziridwa ndi PCR (mkuyu S2).Mapangidwe onse amtundu wa jini adalolanso kuphatikizika kwa atolankhani a Specc11-lacZ atagwidwa.Chifukwa chake, mawu a lacZ otsimikiziridwa ndi madontho a X-gal adagwiritsidwa ntchito ngati chizindikiro cha Specc11.Ma alleles onse adawonetsa mawonekedwe ofanana a lacZ, ndi msampha wamtundu wa DTM096 mu intron 1 wowonetsa mawu amphamvu kuposa RRH048 mu intron 15 (osawonetsedwa).Komabe, Specc1l imasonyezedwa kwambiri, ndi mawu amphamvu kwambiri m'mitsempha ya neural pa E8.5 (Chithunzi 4B), mu neural chubu ndi nkhope pa E9.5 ndi E10.5 (Chithunzi 4C, D), ndi kukula kwa miyendo. ku e10.5 ndi maso (Chithunzi 4D).Tidanenapo kale kuti mawu a SPECC1L mu gawo loyamba la pharyngeal ku E10.5 analipo mu epithelium ndi mesenchyme18, mogwirizana ndi mzere wa CNCC.Kuti tiyese mawu a SPECC1L mu CNCC, tinachita E8.5 neural folds (Chithunzi 4E-J) ndi E9.5 zigawo za chigaza (Chithunzi 4K-).Pa E8.5, SPECC1L yodetsedwa ndi neural folds kwambiri (Mkuyu 4E, H), kuphatikizapo maselo odetsedwa ndi zizindikiro za NCC (Mkuyu 4G, J).Pa E9.5, SPECC1L (mkuyu. 4K, N) mwamphamvu odetsedwa kusamuka CNCC co-zambiri AP2A (mkuyu 4L, M) kapena SOX10 (mkuyu 4O, P).
(A) Chifaniziro cha jini ya mbewa ya Specc11 yomwe ikuwonetsa kuyika kwa vector mu ES DTM096 (intron 1) ndi RRH048 (intron 15) ma cell clones.(BD) lacZ kudetsa kwa heterozygous Specc1lDTM096 miluza yoimira Specc1l mawu kuchokera E8.5 mpaka E10.5.NE = neuroectoderm, NF = neural fold, PA1 = pharyngeal arch yoyamba.(EP) SPECC1L immunostaining ndi NCC markers AP2A ndi SOX10 mu E8.5 (NF; EJ) neural folds ndi E9.5 (KP) zigawo za chigaza.Kudetsa kwa SPECC1L kunkawonedwa kwambiri m'makwinya a neural E8.5 (E, H; mitu ya mivi), kuphatikizapo maselo olembedwa ndi AP2A (F, G; arrowheads) ndi SOX10 (I, J; arrowheads).Pa E9.5, SPECC1L ma CNCC odetsedwa kwambiri (K, N; mivi) olembedwa AP2A (L, M; mivi) ndi SOX10 (O, P; mivi).
Kuwoloka pakati pa heterozygous Specc1lDTM096/+ ndi Specc1lRRH048/+ mbewa kumasonyeza kuti ma gene trap alleles saphatikizana komanso kuti ma heterozygotes ndi ma embryonic homozygotes a jini trap allele amapha embryonic (Table S1).Chiwerengero cha Mendelian chinasonyeza kuchepa kwa moyo wa heterozygotes pa kubadwa (kuyembekezeredwa 1.34 vs. 2.0).Tinaona otsika perinatal imfa pakati heterozygotes, ena anali craniofacial anomalies (mkuyu. S3).Komabe, kutsika pang'ono kwa ma perinatal craniofacial phenotypes kumapangitsa kuti zikhale zovuta kuphunzira njira zawo zapathophysiological.Chifukwa chake, tidayang'ana kwambiri za embryonic lethal phenotype ya homozygous Specc11 mutants.
Mitundu yambiri ya heterozygous kapena homozygous Specc1lDTM096/RRH048 mutant mazira sanayambe pambuyo pa E9.5-10.5 (Mkuyu 5A-D), ndipo neural chubu sichinatseke kutsogolo (Fig. 5B, D) ndipo nthawi zina kutsekedwa kumbuyo (osasonyezedwa) ..Chilema ichi cha cranial neural chubu kutsekedwa chinali chogwirizana ndi ambiri a CNCC olembedwa DLX2 otsalira mu neural folds pa E10.5, kusonyeza kuti palibe dissection (Chithunzi 5A'-D').Kuti tidziwe ngati kukula konse kwa CNCC kunachepetsedwanso, tidalemba mizere ya CNCC ndi GFP m'mizere yathu yamtundu wa jini ndi Wnt1-Cre ndi ROSAmTmG.Timayendetsa GFP+ NCC yosankhidwa ndi GFP- (RFP+) osati NCC kuchokera ku mazira athunthu.Pa E9.5, chiwerengero cha ma CNCC olembedwa ndi GFP otuluka sichinasinthe kwambiri pakati pa WT ndi mazira osasinthika (osasonyezedwa), kusonyeza mafotokozedwe a CNCC.Chifukwa chake, tidayerekeza kuti zotsalira za Wnt1-Cre ndi DLX2 zotsalira m'mikwingwirima yowonekera (Chithunzi 5B') zidachitika chifukwa cha kusanjika kwa CNCC, mwina chifukwa chakuchulukirachulukira kapena kubalalika kwa ma cell a AJ, monga momwe amawonera m'maselo a SPECC1L-kd.Tinagwiritsa ntchito zizindikiro za NCC SOX10, AP2A, ndi DLX2 kutsimikizira kukhalapo kwa CNCC mu neural fold (Chithunzi 5E-R).Pa E8.5, neural fold staining kwa zolembera zonse zitatu za NCC zidawonedwa m'magawo a WT (Fig. 5E, G, I) ndi Specc1l mutant (Fig. 5F, H, J).Pa E9.5, pamene zolembera za NCC zidasokoneza NCC zosamukira m'magawo a WT (mkuyu 5M, O, Q), zotsalira za NCC zotsalira zidawonedwa mumikwingwirima yowonekera ya Specc1l mutant miluza (Mkuyu 5N, P, R).Chifukwa SOX10 ndi DLX2 chizindikiro chosamukira CNCCs, zotsatirazi zikusonyeza kuti SPECC1L-opereŵera CNCCs kukwaniritsa mfundo pambuyo kusamuka koma amalephera kusamuka kuchokera neural folds.
Kuperewera kwa Specc11 kumabweretsa kutsekeka kwa neural chubu, delamination ya cranial neural crest cell ndi ma AJs.
(A, B') E9.5 WT (A) Mwana wosabadwayo atanyamula ma cell a cranial neural crest cell (CNCC) olembedwa ndi Wnt1-Cre (A').Mosiyana ndi izi, Specc11 mutant miluza imawonetsa mikwingwirima yotseguka (B), mitu ya mivi) ndi ma CNCC omwe sanasamuke (B', mitu ya mivi).(C, D') Zithunzi zowala bwino (C, D') ndi immunostaining (C', D') za chikhomo cha CNCC DLX2 cha E10.5 WT miluza (C, C') ndi Specc1l (D, D').Mu ma dzira a WT E10.5, DLX2-positive CNCC imayang'anira ma gill arches (C', mivi), pomwe muzosintha, madontho owoneka bwino amapitilira mumitsempha yotseguka ya neural (D', mivi) komanso m'makona oyamba a pharyngeal (D', mivi).) yokhala ndi madontho (mivi) yosonyeza kusakhazikika bwino komanso kusamuka kwa CNCC.ER) Magawo a WT ndi Specc1l miluza yosinthika pamagawo E8.5 (E-L) ndi E9.5 (M-R) adalembedwa ndi zolembera za NCC SOX10 (E, F, M, N), AP2A (G, H, O, P ) ndi DLX2 (I, J, Q, R).Pa E8.5, madontho a NCC adawonedwa mumtundu wamtundu wa neural fold (NF) ndi magawo osinthika.Co-staining ya SOX10 ndi β-catenin mu E8.5 WT (K) ndi mutant (L) adawonetsa kuwonjezeka kwa β-catenin pamalire a cell mu neural folds.Pa E9.5, madontho akutchire a CNCC osamukira (M, O, Q) adawonedwa, pomwe mu mutants, ma CNCC osasinthika amapaka mikwingwirima yotseguka (N, P, R).(S-Z) Mu vivo AJ kusanthula zolemba m'magawo a coronal a WT ndi Specc11DTM096/RRH048 miluza yokhala ndi kusintha kwa E9.5.Chigawo cha ndege chikuwonetsedwa pakona yakumanja yakumanja.M'zigawo za minyewa yosinthika, mawanga ochulukirapo a F-actin (S, T) ndi myosin IIb (U, V) adawonedwa.Zofanana ndi zotsatira za in vitro mu Mkuyu 3, mu mazira osinthika, kutsekemera kwa membrane kwa β-catenin (W, X) ndi E-cadherin (Y, Z) kunawonedwa.(AA-BB) Ma electron micrograph a gawo la mluza wa kuthengo womwe umayang'ana kupyola malire a cell apical-basal cell amawonetsa chigawo chowoneka bwino cha ma elekitironi chosonyeza zomatira (AA, mivi).Mosiyana ndi izi, m'magawo a Specc11 mutant embryos (BB, mivi), mphambano yonse ya apicobasal ndi electron dense, kusonyeza kuchulukitsidwa kowonjezereka ndi kubalalitsidwa kwa zomatira.
Kuti tiyese malingaliro athu oti kuchepetsa kusanjikiza kumachitika chifukwa cha kusintha kwa AJ, tinayang'ana zolemba za AJ m'mikwingwirima yowonekera ya Specc1l mutant embryos (Mkuyu 5S-Z).Tinawona kuwonjezeka kwa actin stress fibers (Fig. 5S, T) ndi concomitant kuwonjezeka kutanthauzira kwa myosin IIB kudetsa pa actin ulusi (Fig. 5U, V).Chofunika kwambiri, tinawona kuwonjezereka kwa β-catenin (Mkuyu 5W, X) ndi E-cadherin (Mkuyu 5Y, Z) pamalire a intercellular.Tinayang'ananso madontho a β-catenin a NCC mu ma neural folds a E8.5 embryos (Mkuyu 5K, L).Kudetsa kwa β-catenin kumawoneka kolimba mu Specc1l mutant neural folds (Mkuyu 5L ndi K), kutanthauza kuti kusintha kwa AJ kunayamba.Mu ma electron micrographs a zigawo za chigaza cha E9.5 mazira, tinawonanso kuwonjezeka kwa ma electron-dense madontho mu Specc1l mutant miluza poyerekeza ndi WT (Mkuyu 5AA, BB ndi S1E-H).Kuphatikizidwa pamodzi, zotsatirazi zimathandizira zotsatira zathu za in vitro m'maselo a SPECC1L-kd U2OS ndikuwonetsa kuti madontho a AJ amatsogola kukhazikika kwa CNCC m'miluza yathu yosinthika.
Poganizira za ubale wotsutsa womwe umadziwika pakati pa ntchito ya AKT ndi kukhazikika kwa E-cadherin, 17,28 tidayerekeza kukhudzidwa kwa PI3K-AKT siginecha.Komanso, tinaona matuza subepidermal ena mutant mazira amene anathawa kupha (<5%) pa E9.5-10.5 ndipo m'malo anakhazikika padziko E13.5 (mkuyu. S3).Ma Subepidermal vesicles ndi chizindikiro cha kuchepa kwa PI3K-AKT kutengera PDGFRα12.Fantauzzo et al.(2014) inanena kuti kusokonezeka kwa PDGFRα-based PI3K activation mu PdgfraPI3K/PI3K mutant embryos kumabweretsa ma subepidermal vesicles, neural tube defects, ndi cleft palate phenotypes.Zoonadi, milingo ya pan-AKT ndi yogwira phosphorylated Ser473-AKT inachepetsedwa mu vivo mu Specc1l mutant tissues ku E9.5 embryonic kumangidwa (Mkuyu 6A-D).Kuchepa kwa phosphorylated Ser473-AKT kungakhale kwathunthu chifukwa cha kuchepa kwa pan-AKT mu vivo (FIG. 6E) ndi mu vitro (FIG. 6F).Kuchepa kwa in vitro kunawonedwa pokhapokha ma cell a U2OS anali ogwirizana kwambiri ndi kusintha kwa mawonekedwe a cell ndi kachulukidwe ka AJ (Chithunzi 6D).Chifukwa chake, deta yathu ikuwonetsa kuti SPECC1L ndiwowongolera wabwino wa PI3K-AKT siginecha mu craniofacial morphogenesis.
(A-E) E8.5 (A,B) ndi E9.5 (C,D) zigawo za chigaza kapena E9.5 lysates kuchokera ku Specc1l mutant embryos (E) kusonyeza milingo ya phosphorylated yogwira S473-AKT ndi pan-AKT kuchepetsa mapuloteni , poyerekeza ndi kulamulira WT.Kuphulika kwa Kumadzulo kunkachitika pamtundu wamtundu wa lysates ndi mutant lysates pansi pamikhalidwe yomweyi.Zithunzi zowonetsedwa za SPECC1L zidatengedwa kuchokera pachitsamba chimodzi.Chotupa chomwecho chinachotsedwa ndikuwunikidwanso ndi anti-pan-ACT ndi β-actin antibodies.Pan-AKT mu E8.5 neural folds (A, B) ndi milingo ya phosphorylated S473-AKT mu E9.5 zigawo za chigaza zinachepetsedwa kwambiri.(F) Miyezo ya Pan-AKT inachepetsedwa mofananamo mu ma lysates a SPECC1L-kd U2OS maselo omwe amakololedwa pamgwirizano waukulu.Mipiringidzo yolakwika imayimira ma SEM kuchokera kuzinthu zitatu zodziyimira pawokha zaku Western blot.(GJ) Magawo a miluza ya WT pa E9.5 yodetsedwa ndi KI67 ndi caspase 3 yong'ambika, motsatana, kuwonetsa kuchuluka kwa ma cell (G, G') ndi zochitika zazing'ono za apoptotic (H, H').Mimba yosinthika ya Specc11 imawonetsa kuchulukana kwa ma cell (I), koma kuchuluka kwa ma cell omwe akudwala apoptosis kumawonjezeka kwambiri (J).
Kenako tinayang'ana zolembera za kuchuluka ndi apoptosis.Sitinawone kusiyana kulikonse pakukula kwa mazira a E9.5 (Mkuyu 6E, G poyerekeza ndi I) ndi chiwerengero cha 82.5% cha WT mutants ndi 86.5% ya Specc1l mutants yoyesedwa ndi KI67 staining (p <0.56, Fisher's mayeso enieni).Mofananamo, sitinawone kusiyana kulikonse mu apoptosis yoyezedwa ndi kudetsa kwa caspase 3 yodulidwa mu makwinya a neural pa E8.5 mpaka kumangidwa kwa mwana wosabadwayo (osasonyezedwa) (osasonyezedwa).Mosiyana ndi zimenezi, apoptosis inawonjezeka kwambiri mu mazira onse a E9.5 mutant (Mkuyu 6F, H ndi J).Kuwonjezeka konseku kwa apoptosis kumagwirizana ndi kuchepa kwa PI3K-AKT komanso kupha kwa embryonic29,30,31.
Chotsatira, kuti titsimikizire udindo wochititsa kuti PI3K-AKT iwonetsedwe mu kusintha kwa AJ m'maselo athu a kd, tinasintha njira yolamulira ndi kd maselo (Chithunzi 7A-F).Tidagwiritsa ntchito ngati chizindikiritso cha kusintha kwa mawonekedwe amtundu wa phenotype wowonedwa m'maselo olumikizana a SPECC1L-kd, omwe tidawawerengera pogwiritsa ntchito chiyerekezo chautali kwambiri (kutalika) mpaka kumtunda kofanana (m'lifupi).Chiŵerengero cha 1 chikuyembekezeka kwa maselo ozungulira kapena a cuboidal (Chithunzi 7G).Kuwonjezera pa mawonekedwe a selo, tinatsimikiziranso zotsatira za AJ ndi β-catenin staining (Mkuyu 7A'-F').Kuletsa njira ya PI3K-AKT pogwiritsa ntchito wortmannin kunali kokwanira kusintha mawonekedwe a selo m'maselo olamulira (Chithunzi 7A, C) ndi AJ (Chithunzi 7A ').PI3K-AKT activator SC-79 sinakhudze mawonekedwe a cell (FIG. 7A, E) kapena kukulitsa kwa AJ (FIG. 7A') m'maselo owongolera.M'maselo a SPECC1L-kd, kuponderezedwa kwina kwa njira ya PI3K-AKT kunapangitsa kuti apoptosis achuluke (Mkuyu 7B, D) komanso kuwonjezeka kwa β-catenin staining (Fig. 7B '), mogwirizana ndi kusintha kwathu kwa vivo heavy.Chofunika kwambiri, kutsegula kwa njira ya PI3K-AKT kunasintha kwambiri mawonekedwe a selo (Chithunzi 7B, F) ndi AJ phenotypes (Chithunzi 7B").Kusintha kwa mawonekedwe a maselo kunayesedwa ngati chiwerengero cha maselo ozungulira (CCR) ndikufanizira kufunikira monga tafotokozera pamwambapa (FIG. 7G).Zoonadi, m'maselo olamulira (Mkuyu 7G, CCR = 1.56), chithandizo cha wortmannin chinali chokwanira kusintha kwambiri mawonekedwe a selo (Mkuyu 7G, CCR = 3.61, p <2.4 × 10-9) mofanana ndi momwe anawonera. ku SPECC1L.-kd maselo (mkuyu 7G, CCR = 3.46).Kuchiza kwa Wortmannin kwa maselo a SPECC1L-kd (Mkuyu 7G, CCR = 3.60, wosasamala) sizinali zofunikira kwambiri kuposa maselo a kd osagwiritsidwa ntchito (Mkuyu 7G, CCR = 3.46, wosasamala) kapena maselo olamulira opangidwa ndi wortmannin (Mkuyu 7G)., CCR = 3.46, yosasamala) imakhudzanso kutalika kwa selo (7G, CCR = 3.61, yosasamala).Chofunika kwambiri, SC-79 AKT activator inabwezeretsa phenotype ya elongated ya SPECC1L-kd maselo (Mkuyu 7G, CCR = 1.74, p <6.2 × 10-12).Zotsatirazi zimatsimikizira kuti SPECC1L imayang'anira chizindikiro cha PI3K-AKT ndikuwonetsa kuti kuchepa kwapang'onopang'ono kwa SPECC1L kumakhudza kugwirizanitsa kwa selo, pamene kuchepa kwakukulu kumayambitsa apoptosis (Mkuyu 8).
(A-F') Control (A, C, E) ndi SPECC1L-kd (B, D, F) maselo othandizidwa ndi PI3K-AKT pathway inhibitor wortmannin (C, D) kapena SC-79 activator (E, F) Chithandizo .Maselo osagwiritsidwa ntchito osagwiritsidwa ntchito ndi cuboidal (A) okhala ndi β-cat cellular staining (A'), pamene kd maselo amatalikitsidwa (B) ndi kuchuluka kwa β-cat staining (B').Pambuyo pa kuponderezedwa kwa njira ya PI3K-AKT, maselo olamulira amatalikitsidwa (C) ndi β-cat expansion (C'), pamene kd maselo anayamba kuchitidwa apoptosis (D), mofanana ndi mazira athu osinthika kwambiri ndikuwonetsa kwambiri β-paka.kuwononga (D').Pambuyo poyambitsa njira ya PI3K-AKT, ma cell owongolera adakhalabe a cuboidal (E) ndipo amakhala ndi madontho abwinobwino a β-cat (E'), pomwe ma cell a kd amawonetsa mawonekedwe a cell (F) ndi β-cat (F'), kuwonetsa (G) Madigiri a kusintha kwa mawonekedwe a cell mu (AF) adayesedwa pogwiritsa ntchito chiŵerengero cha ma cell roundness (CCR) chautali kwambiri (utali) ndi dimension yofananira (m'lifupi) pogwiritsa ntchito pulogalamu ya MetaMorph.Maselo osagwiritsidwa ntchito (NT) SPECC1L-kd (CCR = 3.46) anali otalika kwambiri kuposa maselo olamulira (CCR = 1.56, p <6.1 × 10-13).Kuletsa kwa Wort kwa njira ya PI3K-AKT m'maselo olamulira kunali kokwanira kuchititsa kutalika kofanana mu mawonekedwe a selo (CCR = 3.61, p<2.4 × 10-9).Mofananamo, kuyambitsa kwa AKT ndi SC-79 m'maselo a SPECC1L-kd kubwezeretsedwa kwa maselo kuti azilamulira (CCR = 1.74, p <6.2 × 10-12).Chithandizo cha Wortmannin cha maselo a SPECC1L-kd chinapangitsa kuti apoptosis achuluke koma palibe kuwonjezeka kwa kusintha kwa mawonekedwe a selo (CCR = 3.60) poyerekeza ndi kd yosasamalidwa (CCR = 3.46, ns) kapena maselo olamulira a wortmannin (3.61) omwe amawonedwa mu .ns = zilibe kanthu.+/- Miyezo ya SEM yama cell 50 ikuwonetsedwa.Kusiyana kwa ziwerengero kudawerengedwa pogwiritsa ntchito t-test ya Student.
(A) Kuyimilira kwadongosolo koletsa ndi kuyambitsa njira ya PI3K-AKT yomwe imapangitsa kusintha kwa AJ ndikupulumutsa, motsatana.(B) Chitsanzo chokonzekera chokhazikika cha mapuloteni a AKT ndi SPECC1L.
Premigratory CNCCs amafuna AJ lysis kupatukana ndi anterior neural fold neuroepithelial cells1,15,32.Kuchulukitsa kwa zigawo za AJ ndi kutayika kwa apical-basal AJ asymmetric kugawa m'maselo osowa a SPECC1L onse mu vitro ndi mu vivo, kuphatikizapo kuyandikira kwa thupi kwa SPECC1L kwa β-catenin, zikusonyeza kuti SPECC1L imagwira ntchito kuti ikhalebe yokhazikika ya AJ. minyewa ya bungwe.actin cytoskeleton.Kugwirizana kwa SPECC1L ndi actin cytoskeleton ndi β-catenin komanso kuwonjezeka kwa chiwerengero cha condensed actin filaments popanda SPECC1L kumagwirizana ndi kuwonjezeka kwa AJ.Kuthekera kwina ndikuti kuchuluka kwa ma actin fibers m'maselo osowa a SPECC1L kumabweretsa kusintha kwa kupsinjika kwapakati.Chifukwa chakuti kupanikizika kwa ma cell kumakhudza mphamvu za AJ 33, kusintha kwa magetsi kungayambitse kufalikira kwa AJ 34.Chifukwa chake kusintha kulikonse kudzakhudza zigawo za CNCC.
Wnt1 imawonetsedwa m'mitsempha yoyambirira ya neural yomwe imayambitsa ma cell a neural crest.Chifukwa chake, Wnt1-cre mizere yolondolera imayika NCC35 isanachitike komanso kusamuka.Komabe, Wnt1 imawonetsanso ma clones a dorsal muubongo omwe amachokera ku makwinya oyambilira a neural 35,36, zomwe zimapangitsa kuti zitheke kuti madontho athu a E9.5 mutants a Wnt1 ma marker mu neural folds otseguka si CNCC.Madontho athu abwino a zolembera za NCC AP2A ndi SOX10 adatsimikizira kuti makwinya owonekera a Specc11 mutant miluza analidi ndi CNCC.Kuonjezera apo, popeza AP2A ndi SOX10 ndi zizindikiro za NCC yosamukira koyambirira, kudetsa kwabwino kumasonyeza kuti maselowa ndi CNCC omwe amasamuka omwe sangathe kutsatiridwa ndi E9.5.
Zambiri zathu zikuwonetsa kuti kuwongolera kwa maselo a AJ ndi SPECC1L kumayanjanitsidwa ndi siginecha ya PI3K-AKT.Chizindikiro cha AKT chimachepetsedwa m'maselo osowa a SPECC1L ndi minofu.Zotsatira za Fantauzzo et al.thandizirani gawo lachindunji la kusaina kwa PI3K-AKT mu craniofacial morphogenesis.(2014) inasonyeza kuti kusowa kwa PDGFRα-based PI3K-AKT siginecha kumabweretsa cleft palate phenotype.Tikuwonetsanso kuti kuletsa njira ya PI3K-AKT ndikokwanira kusintha AJ ndi mawonekedwe a cell m'maselo a U2OS.Mogwirizana ndi zomwe tapeza, Kaini et al.37 inasonyeza kuti kutsika kwa PI3K α110 subunit m'maselo a endothelial kumabweretsa kuwonjezeka kofanana kwa pericellular β-catenin staining, yomwe imatchedwa kuwonjezeka kwa "kulumikizana index".Komabe, m'maselo a endothelial omwe ma actin filaments ali okonzeka kale kwambiri, kuponderezedwa kwa njira ya PI3K-AKT kumabweretsa mawonekedwe a selo lotayirira.Mosiyana ndi izi, ma cell a SPECC1L-kd U2OS adawonetsa mawonekedwe a cell ataliatali.Kusiyanaku kumatha kukhala kwamtundu wa cell.Ngakhale kuponderezedwa kwa PI3K-AKT siginecha kumakhudza kwamuyaya actin cytoskeleton, zotsatira zake pa mawonekedwe a cell zimatsimikiziridwa ndi kusintha kwamphamvu komwe kumachitika chifukwa cha kusintha kwa kachulukidwe ndi kapangidwe ka ulusi wapakati wa actin.M'maselo a U2OS, tidagwiritsa ntchito kusintha kwa mawonekedwe a cell monga chizindikiro cha kusintha kwa SPECC1L-deficient AJ ndi kuchira.Pomaliza, timaganiza kuti kulepheretsa njira ya AKT mu kuchepa kwa SPECC1L kumawonjezera kukhazikika kwa AJ ndikuchepetsa kusokonezeka mu CNCC.
Chosangalatsa ndichakuti, milingo ya pan-AKT idachepetsedwa mu vitro komanso mu vivo kuphatikiza ma phosphorylated 473-AKT pakalibe SPECC1L, kutanthauza kuwongolera kuwonetsa PI3K-AKT pamlingo wa kukhazikika kwa protein ya AKT kapena kubweza.Ma jini a SPECC1L ndi MID1, onse okhudzana ndi matenda a Opitz / GBBB, amalowetsa mapuloteni omwe amakhazikitsa ma microtubules 18,22.Njira yomwe SPECC1L ndi MID1 imagwirizanitsa kukhazikika kwa microtubule sikumveka bwino.Pankhani ya SPECC1L, kukhazikika uku kumaphatikizapo kuwonjezereka kwa acetylation ya kagawo kakang'ono ka microtubules 18.N'zotheka kuti SPECC1L imagwiritsa ntchito njira yofanana kuti ikhazikitse mapuloteni ena monga AKT.Zasonyezedwa kuti acetylation ya zotsalira za lysine mu mapuloteni a AKT kumabweretsa kuchepa kwa membrane localization ndi phosphorylation38.Kuphatikiza apo, kufalikira kwa unyolo wa K63 pamalo otsalira a lysine pa AKT kumafunika kuti kaphatikizidwe kake kawonekedwe ndi kuyambitsa39,40.Zina mwazinthu zingapo zomwe zimagwirizana ndi mapuloteni a SPECC1L omwe amadziwika mumitundu yosiyanasiyana ya yisiti yamitundu iwiri yosakanizidwa, zinayi - CCDC841, ECM2942, APC ndi UBE2I43 - zakhala zikukhudzidwa ndi kubweza kwa mapuloteni kapena kukhazikika kudzera paliponse kapena kutulutsa.SPECC1L ikhoza kutenga nawo gawo pakumasulira pambuyo pomasulira kwa zotsalira za AKT lysine, zomwe zimakhudza kukhazikika kwa AKT.Komabe, gawo lofunika kwambiri la SPECC1L pakukhazikika komanso kukhazikika kwa mapuloteni a AKT siliyenera kufotokozedwa.
Kuwonongeka kwakukulu mu mawonekedwe a SPECC1L mu vivo kudapangitsa kuti chizindikiritso cha AJ chiwonjezeke ndikukuta kwa CNCC kolakwika, komanso kuchuluka kwa apoptosis ndi kupha koyambirira kwa embryonic.Malipoti am'mbuyomu awonetsa kuti kusintha kwa mbewa ndi kuchuluka kwa apoptosis kumalumikizidwa ndi neural tube defects 44,45,46,47 ndi craniofacial defects48.Zanenedwa kuti kufa kwa maselo ochulukirapo m'mitsempha ya neural kapena pharyngeal arches kungapangitse chiwerengero chosakwanira cha maselo ofunikira kuti ayendetse bwino morphogenetic 48,49,50.Mosiyana ndi izi, mizere yathu ya SPECC1L yopanda mphamvu yokhala ndi mawonekedwe ocheperako a SPECC1L imangowonetsa kusintha kwa AJ popanda umboni wa kufa kwa cell.Komabe, kuletsa kwamankhwala kwa njira ya PI3K-AKT m'maselo a Kd awa kunapangitsa kuti apoptosis achuluke.Chifukwa chake, kuchepa pang'ono kwa mawu a SPECC1L kapena ntchito kumatsimikizira kupulumuka kwa maselo.Izi zikugwirizana ndi zomwe zinachitikira Specc11 mutant miluza yomwe imathawa kumangidwa ku St.E9.5-mwina chifukwa cha kuchepa kwa jini yogwira bwino-amatha kutseka machubu awo a neural ndikusiya pambuyo pake mu chitukuko, nthawi zambiri ndi zolakwika za craniofacial (mkuyu S3).Zomwe zimagwirizana ndi izi ndizochitika kawirikawiri za mazira a heterozygous Specc1l omwe ali ndi vuto la craniofacial-mwina chifukwa cha kuchuluka kwa kugwidwa kwa majini-komanso kupezeka kwa zebrafish momwe imodzi mwa ma orthologues awiri a SPECC1L (specc1lb) imayambitsa ma embryonic phenotypes, kuphatikizapo kutaya kwa nsagwada za m'munsi ndi ming'alu ya mayiko awiri51.Choncho, kusintha kwa heterozygous SPECC1L kutayika kwa ntchito komwe kumazindikiridwa mwa odwala aumunthu kungayambitse kuwonongeka kochepa mu ntchito ya SPECC1L panthawi ya craniofacial morphogenesis, yokwanira kufotokozera ming'alu yawo ya orofacial.SPECC1L-based regulation of intercellular contacts ingathandizenso palatogenesis ndi fusion ya pharyngeal arches.Kufufuza kwina kwa ntchito ya SPECC1L kudzathandiza kufotokozera momwe kulumikizana kwakanthawi kochepa mu CNCC kutsekedwa kwa neural chubu mu neuroepithelial cell motility ndi craniofacial morphogenesis.
U2OS osteosarcoma control ndi SPECC1L-kd maselo afotokozedwa kale (Saadi et al., 2011).Ma antibodies motsutsana ndi SPECC1L adadziwikanso kale (Saadi et al., 2011).Anti-β-catenin antibodies (kalulu; 1: 1000; Santa Cruz, Dallas, TX) (mbewa; 1: 1000; Cell Signaling Technology, Danvers, MA), myosin IIb (1: 1000; Sigma-Aldrich, St. Louis) ) , MO) ), E-cadherin (1:1000; Abkam, Cambridge, MA), AP2A (1:1000; Novus Biologicals, Littleton, Colo.), SOX10 (1:1000; 1000; Aviva Systems Biology, San Diego , California), DLX2 (1:1000; Abcam, Cambridge, MA), phospho-Ser473-AKT (1:1000; Cell Signaling Technology, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA ) , KI67 (1: 1000; Cell Signaling Technology, Danvers, MA), cleaved caspase 3 (1: 1000; Cell Signaling Technology, Danvers, MA) ndi β-actin (1: 2500; Sigma-Aldrich, St. Louis, MO ) idagwiritsidwa ntchito monga momwe tafotokozera..Actin filaments anali odetsedwa ndi Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Maselo owongolera a U2OS ndi ma cell a SPECC1L-kd adakulitsidwa mu DMEM ya shuga wambiri wophatikizidwa ndi 10% fetal bovine serum (Life Technologies, Carlsbad, CA).Kwa kusintha kwa AJ, maselo a 2 x 105 adayikidwa pa galasi lopangidwa ndi 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) ndikuwona kusintha kwa mawonekedwe a selo.Maselo anasonkhanitsidwa pazigawo zosiyanasiyana za nthawi: maola 4 mutabzala (t = 1), maola 24 mutabzala (t = 2), kugwirizana popanda kusintha kwa mawonekedwe a selo (t = 3), kusintha kwa mawonekedwe a selo (t = 4) , 24 h pambuyo pa kusintha kwa mawonekedwe a selo (t = 5) ndi 48 h pambuyo pa kusintha kwa mawonekedwe a selo (t = 6) (Mkuyu 1, 2, 3).Kuti musinthe njira ya PI3K-AKT, ma cell adakulitsidwa pazigawo zomwe zawonetsedwa ndi PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) kapena SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Sing'anga yokhala ndi mankhwalawo idasinthidwa tsiku lililonse.
Zojambula za chimango ndi chimango zidapangidwa paziwongolero zamoyo ndi ma cell a KD pansi pazikhalidwe zachikhalidwe, ndipo zithunzi zosiyanitsa zimasonkhanitsidwa mphindi 10 zilizonse kwa masiku 7.Zithunzi zinapezedwa pogwiritsa ntchito makina oonera pakompyuta a Leica DM IRB okhala ndi siteji ya makina komanso cholinga cha 10 × N-PLAN cholumikizidwa ndi kamera ya QImaging Retiga-SRV.Pakujambula, zikhalidwe zama cell zidasungidwa pa 37 ° C mumlengalenga wonyowa ndi 5% CO2.
Mizere iwiri yamtundu wa ES cell trap DTM096 ndi RRH048 yochokera ku Regional Mutant Mouse Resource Center (UC Davis, CA) idagwiritsidwa ntchito popanga mizere ya mbewa yoperewera ya Specc11, yosankhidwa Specc1lgtDTM096 ndi Specc1lgtRRH046.Mwachidule, ma cell a 129/REJ ES anabayidwa mu C57BL6 blastocysts.Makoswe aamuna a chimeric adaberekedwa ndi mbewa zazikazi za C57BL6 kuti zizindikire ana omwe ali ndi mtundu wa malaya a agouti.Kukhalapo kwa ma gene trap vector kunkagwiritsidwa ntchito kuzindikira ma heterozygotes.Mbewa zinasungidwa pamtunda wosakanikirana wa 129 / REJ; C57BL6.Malo a malo oyikapo ma genetic trap vector adatsimikiziridwa ndi RT-PCR, kutsatizana kwa ma genome, ndi genetic complementation (Supplementary Figure 1).Kufufuza mzere wa CNCC wa mbewa ziwiri za heterozygous Specc1lGT, ROSAmTmG (#007576) ndi Wnt1-Cre (#003829) mbewa (Jackson Laboratory, Bar Harbor, ME) adawoloka kuti apange ROSAmTmG ndi Wnt1-Cre allele mu Specc1l mutant embryos.Kuyesera konse kwa mbewa kunachitika motsatira ndondomeko zovomerezedwa ndi Institutional Animal Care and Use Committee ya University of Kansas Medical Center.
Mimba inayikidwa mu (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) kwa mphindi 60 kutentha.Pambuyo pokonza mu X-gal staining solution (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg/ml X-gal) Kukula kwa madontho kunachitika pa 37°C .°C mkati mwa maola 1-6.Miluza idakhazikitsidwa pambuyo pa 4% PFA ndikuwonera.
Kwa Western blotting, maselo anali lysed mu passive lysis buffer (Promega, Fitchburg, WI) kuwonjezeredwa ndi chisakanizo cha HALT protease inhibitors (Sigma-Aldrich, St. Louis, MO).Ma Lysates adakonzedwa pa 12% polyacrylamide Mini-PROTEAN TGX okonzeka ma gels (Bio-Rad, Hercules, CA) ndipo amasamutsidwa ku Immobilon PVDF nembanemba (EMD Millipore, Billerica, MA).Ma membrane adatsekedwa mu mkaka wa 5% mu PBS wokhala ndi 0.1% Tween.Ma antibodies amaikidwa usiku wonse pa 4 ° C kapena kwa ola limodzi kutentha kwa chipinda.Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) idagwiritsidwa ntchito popanga ma siginecha.Pofuna kuteteza chitetezo cha mthupi, mazira amaikidwa usiku umodzi mu 4% PFA/PBS ndi cryopreserved.Mitsempha ya minofu inatsekedwa mu PBS yomwe ili ndi 1% seramu ya mbuzi (Thermo Scientific, Waltham, MA) ndi 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) ndipo kenako incubated pa 4 ° C mu incubator panthawi ya usiku.okhala ndi anti-antibody ndi fluorescent secondary antibody (1:1000) kwa ola limodzi pa 4°C.Zigawo zothimbirira zidayikidwa mu ProLong golide medium (Thermo Scientific, Waltham MA) ndipo zithunzi zosalala zidapezedwa pogwiritsa ntchito microscope ya Leica TCS SPE confocal.Kuteteza chitetezo chilichonse kunachitika ngati kuyesa katatu kodziyimira pawokha pamagulu amitundu iwiri yosinthika.Kuyesera koyimilira kukuwonetsedwa.
Maselo adalowetsedwa mu buffer yosinthidwa ya RIPA (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, ndi HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Mwachidule, ma lysates adayeretsedwa kale ndi mapuloteni a G maginito mikanda (Life Technologies, Carlsbad, CA) ndiyeno amalowetsedwa usiku wonse pa 4 ° C. ndi anti-SPECC1L kapena IgG mapuloteni G mikanda ankagwiritsidwa ntchito pochotsa SPECC1L ndipo kuzunguliridwa kwa Western kunkachitika pogwiritsa ntchito anti -β-catenin antibody yofotokozedwa pamwambapa Mayesero a co-IP omwe akuwonetsedwa akuyimira mayesero anayi odziimira okha.
Maselo okhazikika okhazikika kapena ma embryonic tissues adaperekedwa ku electron microscopy center pa University of Kansas Medical Center.Mwachidule, zitsanzo zinayikidwa mu EMbed 812 resin (Electron Microscopy Sciences, Fort Washington, PA), yopangidwa ndi polymer usiku wonse pa 60 ° C, ndipo inagawidwa pa 80 nm pogwiritsa ntchito Leica UC7 ultramicrotome yokhala ndi tsamba la diamondi.Magawo adawonetsedwa pogwiritsa ntchito maikulosikopu yamagetsi ya JEOL JEM-1400 yokhala ndi mfuti ya 100 kV Lab6.
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