ASTM A790 2507/2205 1.4462 / 1.4410 Duplex Welded Tube Kubikoresho bya chimique yinganda zikora imiti, Kubura SPECC1L bituma habaho ituze ryingingo zifatanije kandi bikagabanuka kumeneka kwingirabuzimafatizo.

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ASTM A790 2507/2205 1.4462 / 1.4410 Duplex Welded Tube Yinganda Zimiti

Liaocheng Sihe SS Ibikoresho Co, Ltd.ni uruganda ruyoboye inzobere mu byuma bidafite ingese, imiyoboro itagira umuyonga, umuyoboro utagira ingano hamwe n'ibindi.Mu rwego rwo korohereza abakiriya, twahinduye imiyoboro n'imiyoboro.Liaocheng Sihe SS Ibikoresho Co, Ltd.ifite ibikoresho bigezweho byo gukora no kugerageza.Turashobora guhaza rwose ibyo usabwa.Ukurikije ibisanzwe rwose, imiyoboro yakozwe natwe ihora ifite kwihanganira OD na WT.Kugenzura kwihanganira gukurikiza rwose gutanga ibipimo.Ibicuruzwa byacu burigihe kunyurwa nabakiriya.Abakiriya baguze ibicuruzwa byacu byungutse byinshi.
a) OD (Diameter yo hanze): 3.18mm kugeza kuri 101,6mm
b) WT (Uburebure bw'urukuta): 0.5mm kugeza 20mm
c) Uburebure: Ukurikije ibyo umukiriya asabwa
d) Ibipimo: ASTM A312;ASTM A269;ASTM A789;ASTM A790 nibindi
e) Uburyo bwo gutunganya: ERW, EFW nibindi

Abashoferi berekana ingingo eshatu kuri slide.Koresha inyuma na buto ikurikira kugirango unyuze kumurongo, cyangwa utubuto twa slide mugenzuzi kugirango urangire unyuze kuri buri slide.
Ingirabuzimafatizo zo mu bwoko bwa Cranial neural crest selile (CNCC) zinyeganyeza ziva mu ntangangore kandi zimukira mu rubingo rwa pharyngeal, zigize igice kinini cyo hagati.Imikorere idahwitse ya CNCC igira uruhare runini muri etiologiya ya orofacial cleft, malformation yavutse.Heterozygous SPECC1L ihindagurika ryabonetse kubarwayi bafite udukoko tudasanzwe na syndromic.Hano, turatanga raporo yongerewe irangi ryibikoresho bya Canonical bifatanye (AJ), cat-catenin na E-cadherin mu ngirabuzimafatizo za SPECC1L zifite umuco, hamwe na micrografi ya electron yerekana ikwirakwizwa rya AJ.Kugirango twumve uruhare rwa SPECC1L muri craniofacial morphogenez, twashizeho icyitegererezo cyimbeba ya Specc1l.Guhindura ibitsina byombi ni urusoro rwica kandi rugaragaza imitsi yangirika no gufunga CNCC.AJ proteine ​​irangi yiyongera muburyo bwimiterere ya mutant.Iyi nenge ya AJ ijyanye nubusembwa bwo gusiba CNCC, bisaba guseswa AJ.Mubyongeyeho, ihinduka rya Specc11 ryagabanije ibimenyetso bya PI3K-AKT kandi byongera apoptose.Muri vitro, kubuza byoroheje PI3K-AKT amarenga mu ngirabuzimafatizo zo mu gasozi byari bihagije kugira ngo AJ ihinduke.Icyangombwa, AJ impinduka zatewe na SPECC1L gukomanga zirashobora guhindurwa no gukora inzira ya PI3K-AKT.Ufatiye hamwe, aya makuru yerekana ko SPECC1L, nkigenzura rishya ryerekana ibimenyetso bya PI3K-AKT hamwe n’ibinyabuzima bya AJ, bisabwa kugirango hafungwe imiyoboro y’imitsi ndetse na CNCC.
Cranial neural crest selile (CNCCs) yegereye dorsal neuroectoderm hanyuma igatandukana na neuroepitelium yimitsi ikura ikura binyuze mubikorwa birimo inzibacyuho ya epiteliyale-mesenchymal (EMT) 1,2,3.Kwimura epithelial CNCCs bihagarika imiyoboro ihuza imitsi hanyuma bigahinduka mesenchymal CNCCs yuzuza imitsi ya mbere na kabiri ya pharyngeal arche kandi igizwe na karitsiye ya craniofacial.Rero, ingirabuzimafatizo zigenga imikorere ya CNCC zikunze guhungabana muri etiologiya ya craniofacial congenital anomalies nka orofacial clefts, bikunze kwibasira 1/800 bavuka muri Amerika yonyine.Imwe mu miterere ivuka8.
Kurandura CNCC bihurirana no gufunga imiyoboro yimbere yimbere hagati yiminsi 8.5 na 9.5 yo gukura kwa misoro mu mbeba.Guhinduranya imbeba zitari nke za orofacial zifitanye isano na gen zigaragaza kandi uburyo bumwe na bumwe bwinenge zifata imitsi, harimo Irf69,10, Ghrl310, Cfl111, na Pdgfrα12.Nyamara, inzira yo gufunga imiyoboro yimitsi no gutondekanya CNCC irashobora gufatwa nkigenga, kubera ko imbeba ya Splotch mutant (Pax3) igaragaza inenge mu gufunga imiyoboro y’imitsi nta ngaruka igira ku miterere ya CNCC cyangwa kwimuka 13,14.Iyindi mbeba yimbeba ifite inenge mugutandukanya CNCC no gufunga imiyoboro ya neural bizafasha gusobanura ishingiro rya molekile rusange yizi nzira zombi.
Kwigunga kwa CNCC muri selile neuroepithelial bisaba gusesa imiyoboro ifatika (AJs), igizwe ninganda za poroteyine zirimo, E-cadherin, β-catenin, α-E-catenin, na α-actinin ifitanye isano na filime ya actin 2 . Ubushakashatsi bukabije E-cadherin mubice byimitsi byerekanaga kugabanuka cyangwa gutinda gusohora CNCC.Ibinyuranye, guhagarika E-cadherin bivamo ibyiciro 15,16.Byinshi mubintu bihuza EMT mugihe cyo gutondekanya CNCC ni ibintu byandikirwa (AP2α, Id2, FOXD3, SNAIL, TWIST, SOX10) hamwe na matrice idasanzwe (ECM) ivugurura poroteyine nka matrix metalloproteinase (MMPs), nyamara CNCCs ni cytoskeletal AJ igenzura. ntaramenyekana.Inzira ya PI3K-AKT izwiho kurwanya urugero rwa E-cadherine, cyane cyane mubushakashatsi bwa kanseri17.Ubushakashatsi buherutse gukorwa bwerekanye ko gutakaza ibimenyetso bya PDGFα bishingiye kuri PI3K-AKT byerekana imbeba biganisha ku miterere idasanzwe ya craniofacial, harimo umunwa wuzuye hamwe nudukoko two mu mitsi12.Ariko, isano iri hagati yinzira ya PI3K-AKT nu mutekano wa AJ kurwego rwa CNCC ntisobanutse.
Twabonye mbere ko SPECC1L ari gene ya mbere ya mutant mu bantu babiri bafite ururenda rukomeye ruva mu kanwa kugeza ku jisho, ruzwi ku izina rya oblique cleft (ObFC) cyangwa Tessier IV18.Ihinduka rya SPECC1L ryagaragaye mu miryango ibiri y’ibisekuru byinshi hamwe na syndrome ya Opitz G / BBB yigenga (OMIM # 145410), aho abantu bagize ingaruka bagaragaje hyperdistance hamwe na lip / palate19, no mu muryango umwe ufite syndrome ya Tibi ikabije (OMIM # 145420) 20 .ibice birenga ica kabiri c'indwara ya syndrome ya Opitz G / BBB ifitanye isano na X (OMIM # 300000) kandi iterwa no guhinduka kwa gene MID1, ikubiyemo poroteyine 22 ya microtubule ifitanye isano na skeleton ya selile.Turakekeranya ko SPECC1L, na poroteyine ifitanye isano na microtubules na actin cytoskeleton, irashobora guhuza ibimenyetso bikenewe kugirango actin cytoskeleton ivugururwe mugihe cyo gufatira hamwe kwimuka 18.Binyuze muri vitro no mubushakashatsi bwa vivo, ubu turasobanura SPECC1L nkigenzura rishya ryimikorere ya AJ binyuze mubimenyetso bya PI3K-AKT.Ku rwego rwa selire, ibura rya SPECC1L ryatumye igabanuka ryurwego rwa poroteyine ya pan-AKT ndetse no kwiyongera kwa apical-basal dispersion ya AJ, byavanyweho no gukoresha imiti ya AKT inzira.Muri vivo, insoro zabuze Specc11 zerekana kwangirika kw'imitsi yo mu mitsi kandi bigabanya gutandukana kwa CNCC.Rero, SPECC1L ikora mumikorere ya selile igenzurwa cyane-yerekana ibimenyetso bisabwa kumikorere isanzwe ya CNCC mugihe cya morfogenezi yo mumaso.
Kugirango tugaragaze uruhare rwa SPECC1L kurwego rwakagari, twakoresheje ibyasobanuwe mbere bihamye osteosarcoma selile selile U2OS ibuze muri SPECC1L18.Utugingo ngengabuzima U2OS duhamye hamwe na SPECC1L (kd) gukubita byagabanutse (60-70%) kugabanuka kurwego rwa transcript na proteine ​​za SPECC1L, hamwe nudusembwa twimuka no kuvugurura actin cytoskeleton 18. Ibinyuranye, kugabanuka gukabije kwigihe gito muri SPECC1L yerekanwe kuganisha ku nenge ya mitoto 23.Tumaze kuranga, twasanze selile zacu zihamye za SPECC1L-kd zahinduye morphologie kurwego rwo hejuru rwo guhuza (Ishusho 1).Utugingo ngengabuzima kugiti cyawe hamwe na kd selile zihurira hamwe byasaga (Ishusho 1A, D).Nyuma yamasaha 24 nyuma yo guhuza, selile igenzura yagumanye imiterere ya cuboidal (Igishusho 1B, E), mugihe selile SPECC1L-kd irambuye (Ishusho 1C, F).Ingano yiyi mpinduka mumiterere ya selile yafashwe na vivo live amashusho ya selile igenzura na kd selile (firime 1).Kugirango tumenye uruhare rwa SPECC1L muri selile zihuza, twabanje gusuzuma imvugo yacyo.Twabonye ko poroteyine ya SPECC1L yiyongereye nyuma yo guhuza (Igicapo 1G), mu gihe urwego rwa transcript rwa SPECC1L rutigeze rwiyongera (Ishusho 1H).Byongeye kandi, uko ubucucike bwakagari bwiyongereye, poroteyine ya SPECC1L yegeranijwe ku mbibi z’imitsi (Igishusho 2A-E), hamwe n’ishusho ihujwe n’iya membrane ifitanye isano na cat-catenine (Ishusho 2A'-E ').Urebye ishyirahamwe rya SPECC1L hamwe na actin cytoskeleton 18,23 twavuze ko SPECC1L ikorana n’imikorere ifatika (AJ).
.Herekanwa hano ni bitatu muri bitandatu byigihe (T1, T3, T6) twahisemo kubucucike butandukanye..Iburengerazuba bwiburengerazuba bwa SPECC1L byerekana umurongo uteganijwe wa kDa 120 hamwe na bande iremereye cyane, birashoboka ko byahinduwe nyuma yubusobanuro (*).Isesengura rya blot yo muburengerazuba ryakozwe mubihe bimwe kugirango habeho guhurira hamwe.Amashusho yerekana SPECC1L kumurongo muto kandi muremure wafatiwe kumurongo umwe.Blot imwe yakuweho hanyuma yongera gusuzumwa na antibody-actin.(H) Isesengura ryinshi rya RT-PCR ryerekanye ko nta mpinduka zikomeye ziri murwego rwa transcript ya SPECC1L.Utubari twibeshya twerekana SEM kuva mubigeragezo bine byigenga.
.Ibice bitanu byambere muribi bihe byashizwemo ingirabuzimafatizo imwe (T1), 50-70% guhuza utugingo ngengabuzima duto (T2), guhuza utabanje kuvugurura kd selile (T3), guhindura kd selile (T4), no guhindura amasaha 24.muburyo bwinyuma bwa kd (T5) selile.Poroteyine ya SPECC1L yari ikwirakwijwe cyane muri cytoplazme kuri T1 (A), ariko kwirundanya kwayo byagaragaye ku mbibi z’imitsi hagati yigihe cyakurikiyeho (B - E, imyambi).(FJ) β-catenin yerekana kwirundanya bisa kumipaka ihuza imipaka ifitanye isano na AJ complex... Byahindutse.Utubari = 10 µm.
Twagerageje kumenya ingaruka zo kubura SPECC1L kuri AJ.Twifashishije ibimenyetso byinshi bifitanye isano na AJ, harimo ibice byemewe bya F-actin, myosin IIb, β-catenin, na E-cadherin 24,25,26,27.Actin stress fibre yiyongereye muri selile SPECC1L-kd nkuko byasobanuwe mbere (Ishusho 3A, B) 18.Myosin IIb ifitanye isano na filime ya actin yerekanye ubwiyongere busa muri selile SPECC1L-kd muri vitro (Ishusho 3C, D).AJ ifitanye isano β-catenin ihuza na cadherine kuri selile ya selile, yerekana uburyo busanzwe bwerekana "ubuki" muburyo bwo kugenzura cubocytes (Ishusho 3E, G).Igishimishije, mumashusho aringaniye ukoresheje microscopi ya confocal, β-catenin (Igishusho 3E, F) na E-cadherin (Igishusho 3G, H) yandujwe kuri selile ya selile ya selile idafite SPECC1L yerekanaga uburyo bugaragara bwo kwanduza kwaguka.Uku kwaguka kwa AJ gufitanye isano β-catenine yanduye muri selile ya kd byagaragaye cyane aho bihurira, ariko byagaragaye ko byabanjirije impinduka mumiterere ya selile (Ishusho 2F-J, F'-J ').Kugirango tumenye imiterere yumubiri yagutse ya AJ, twasuzumye imbibi za selile hejuru yubuso bwibanze bwa selile SPECC1L-kd U2OS dukoresheje microscopi electronique (TEM) (Ishusho 3I, J).Bitandukanye no kugenzura ingirabuzimafatizo (Igishusho 3I), zari zifite uturere twinshi twa elegitoronike twerekana AJ (imyambi), selile kd (Igishusho 3J) yerekanaga uturere twinshi, duhuza uturere twinshi twa elegitoronike twerekana AJ ku ndege ya apicobasal..Mubyongeyeho, ku bice bihinduranya, twarebye ingirabuzimafatizo ngari mu ngirabuzimafatizo za kd (Igicapo S1A, B), gisobanura uburyo bwagutse bwa β-catenin na E-cadherine yerekana amabara (Ishusho 3F, H).Mu rwego rwo gushyigikira uruhare rwa SPECC1L muri AJs, cat-catenin yafatanywe ubudahangarwa na SPECC1L muri lysates ya selile U2OS ihuza (Ishusho 3K).Hamwe nogukingira kwagutse kubimenyetso bya AJ, isesengura rya TEM ryahujwe na hypothesis yacu ko kubura SPECC1L byongera AJ apical-basal density na variance.
(AH) Yiyongereye F-actin irangi muri kd selile mumasaha 48 nyuma yo guhuza (T6; A, B).Guhindura irangi rya myosine IIb ifitanye isano na F-actin (C, D).Uburyo bwiza bwa β-catenin na E-cadherin membrane yanduza ingirabuzimafatizo (E, G) yazamuwe muri selile SPECC1L-kd (F, H).Utubari = 10 µm..Ingirabuzimafatizo zigaragaza uturere dutandukanye twa elegitoronike yerekana guhuza (I, imyambi).Ibinyuranyo, ihuriro rusange rya apical-basal muri selile SPECC1L-kd ryagaragaye cyane kuri elegitoronike (J, imyambi), byerekana ubwinshi bwikwirakwizwa no gukwirakwiza imiyoboro ifatanye..Ishusho yakuwe ahantu hamwe yerekana kimwe mubigeragezo bine byigenga.
Kugira ngo dusobanukirwe uruhare rwa SPECC1L muri morfogenezi ya craniofacial, twashizeho icyitegererezo cyimbeba ya Specc1l ikoresheje imirongo ibiri yigenga ya ES trap selile, DTM096 na RRH048 (BayGenomics, CA), igereranya intron 1 na transcript ya Specc1l yafashwe kuri 15 (Ishusho 1) .4A, ishusho S2).Ikibanza cya genomic cyinjiza uburiganya cyagenwe na genome yose uko yakabaye kandi byemejwe na PCR (Ishusho S2).Ibishushanyo mbonera byombi byemereye kandi guhuza abanyamakuru ba Specc11-lacZ bamaze gufatwa.Kubwibyo, imvugo ya lacZ yagenwe na X-gal irangi yakoreshejwe nkikimenyetso cyerekana imvugo ya Specc11.Alleles zombi zerekanye imvugo ya lacZ isa, hamwe na DTM096 umutego wa gene muri intron 1 yerekana imvugo ikomeye kuruta RRH048 muri intron 15 (iterekanwa).Nyamara, Specc1l iragaragazwa cyane, hamwe nimvugo ikomeye cyane mumyanya mitsi kuri E8.5 (Igicapo 4B), mumitsi ya neural nu nzira yo mumaso kuri E9.5 na E10.5 (Igicapo 4C, D), no mugukuza ingingo. kuri E10.5 n'amaso (Ishusho 4D).Twabanje kuvuga ko imvugo ya SPECC1L mugice cya mbere cya pharyngeal kuri E10.5 yari ihari muri epitelium no munsi ya mesenchyme18, ihuje numurongo wa CNCC.Kugerageza imvugo ya SPECC1L muri CNCC, twakoze E8.5 imitsi (Ishusho 4E-J) na E9.5 ibice bya gihanga (Ishusho 4K-).Kuri E8.5, SPECC1L yandujije imitsi cyane (Ishusho 4E, H), harimo selile zanditseho ibimenyetso bya NCC (Ishusho 4G, J).Kuri E9.5, SPECC1L (Igishusho 4K, N) yanduye cyane CNCC yimuka ifatanije na AP2A (Igishusho 4L, M) cyangwa SOX10 (Ishusho 4O, P).
..NE = neuroectoderm, NF = ububiko bwimitsi, PA1 = ubwambere pharyngeal arch..Irangi rya SPECC1L ryagaragaye cyane mubice by'imitsi E8.5 (E, H; imyambi), harimo selile zanditseho AP2A (F, G; imyambi) na SOX10 (I, J; imyambi).Kuri E9.5, SPECC1L yandujije cyane CNCCs yimuka (K, N; imyambi) yanditseho AP2A (L, M; imyambi) na SOX10 (O, P; imyambi).
Kwambukiranya hagati ya heterozygous Specc1lDTM096 / + na Specc1lRRH048 / + imbeba byerekana ko imitego yombi ya trap alleles ituzuzanya kandi ko ivangitirane rya heterozygote hamwe na homozygote ya emboroon kuri trap allele yica urusoro (Imbonerahamwe S1).Ikigereranyo cya Mendelian cyerekanye ko igabanuka ry’imibereho ya heterozygote ikivuka (biteganijwe 1.34 na 2.0).Twabonye impfu nke za perinatal muri heterozygote, bamwe bari bafite anomalie ya craniofacial (Ishusho S3).Nyamara, kwinjira kwinshi kwa perinatal craniofacial phenotypes bituma bigora kwiga uburyo bwimikorere ya patrophysiologique.Kubwibyo, twibanze kuri embryonic yica phenotype ya homozygous Specc11 mutant.
Imvange nyinshi ziterwa na heterozygous cyangwa homozygous Specc1lDTM096 / RRH048 insoro za mutant ntizigeze zikura nyuma ya E9.5-10-10 (Ishusho 5A - D), kandi umuyoboro w'imitsi ntiwigeze ufunga imbere (Ishusho 5B, D) kandi rimwe na rimwe ufunga inyuma (uterekanwa) ..Iyi nenge yo gufunga imiyoboro ya cranial neural yahujwe na CNCC yaranze DLX2 isigaye mumitsi ya E10.5, byerekana ko itatandukanijwe (Igicapo 5A'-D ').Kugirango tumenye niba ingano rusange ya CNCC nayo yagabanutse, twashushanyije imirongo ya CNCC hamwe na GFP mumirongo yacu ya trap ya gene hamwe na Wnt1-Cre na ROSAmTmG.Dutondekanya GFP + NCC na GFP- (RFP +) itari NCC kuva insoro zose.Kuri E9.5, igipimo cyurutonde rwa GFP cyanditseho CNCCs nticyahindutse cyane hagati ya WT na insoro za mutant (ziterekanwa), byerekana ibisobanuro bisanzwe bya CNCC.Kubwibyo, twavuze ko Wnt1-Cre na DLX2 isigaye yanduye mu mitsi igaragara (Igicapo 5B ') byatewe na CNCC ifite inenge, bishoboka ko byatewe no kwiyongera kwinshi cyangwa gukwirakwiza selile AJ, nkuko bigaragara muri selile SPECC1L-kd.Twifashishije ibimenyetso bya NCC SOX10, AP2A, na DLX2 kugirango twemeze ko CNCC ihari mubice byimitsi (Ishusho 5E-R).Kuri E8.5, ubwonko bwanditseho ibimenyetso byose uko ari bitatu bya NCC byagaragaye mu bice bya WT (Ishusho 5E, G, I) na Specc1l mutant (Ishusho 5F, H, J).Kuri E9.5, mugihe ibimenyetso bya NCC byanditseho NCC yimuka mu bice bya WT (Igicapo 5M, O, Q), irangi rya NCC ryagaragaye mu mitsi y’imitsi igaragara ya insoro ya mutant ya Specc1l (Ishusho 5N, P, R).Kuberako SOX10 na DLX2 biranga kwimuka CNCCs, iki gisubizo cyerekana ko CNCCs yabuze SPECC1L igera kubisobanuro nyuma yo kwimuka ariko ikananirwa kwimuka ikava mumitsi.
Kubura Specc11 biganisha ku gufunga imitsi idafite inenge, gusiba ingirabuzimafatizo ya Cranial neural crest selile na AJs.
(A, B ') E.Ibinyuranye, insoro ya mutant ya spcc11 yerekana gufungura imitsi (B), imyambi) na CNCCs zitimutse (B ', imyambi)..Muri insoro ya WT E10.5, DLX2-nziza ya CNCC ikoronije ingarani ya gill (C ', imyambi), mugihe muri mutant, irangi ryibonekeje riguma mumitsi ifunguye (D', imyambi) no mubitereko byambere bya farynge (D ', imyambi).) hamwe no gusiga (imyambi) byerekana gusiba nabi no kwimuka kwa CNCC.ER) Ibice bya WT na Specc1l insoro za mutant ku cyiciro E8.5 (E - L) na E9.5 (M - R) byanditseho ibimenyetso bya NCC SOX10 (E, F, M, N), AP2A (G, H, O, P) na DLX2 (I, J, Q, R).Kuri E8.5, irangi rya NCC ryagaragaye mubice byo mu gasozi (NF) no mu bice bya mutant.Kwanduza SOX10 na β-catenin muri E8.5 WT (K) na mutant (L) byagaragaje ko β-catenine yiyongereye ku mipaka y’utugingo ngengabuzima.Kuri E9.5, hagaragaye ubwoko bwishyamba bwa CNCCs yimuka (M, O, Q), mugihe muri mutant, CNCC idafite ibyapa byanditseho imitsi (N, P, R)..Indege igereranijwe yerekanwa mugice cyo hejuru cyiburyo.Mu bice by'imitsi ihindagurika, hagaragaye irangi rya F-actin (S, T) na myosine IIb (U, V).Bisa na vitro ibisubizo ku gishushanyo cya 3, mu nsoro za mutant, byongerewe imbaraga zanduza β-catenin (W, X) na E-cadherine (Y, Z) byagaragaye..Ibinyuranye, mu bice bigize insoro ya Specc11 (BB, imyambi), ihuriro ryose rya apicobasal ni ubucucike bwa electron, byerekana ubwinshi bwikwirakwizwa no gukwirakwiza imiyoboro ifatanye.
Kugirango dusuzume hypothesis ko kugabanya ibice biterwa na AJ yahinduwe, twasuzumye ikirango cya AJ mumyanya ndangagitsina ya spcc1l ya misoro ya mutant (Ishusho 5S-Z).Twabonye ubwiyongere bwa fibre yibibazo (Igicapo 5S, T) hamwe no kwiyongera kwa myosine IIB yanduza fibre ya actin (Ishusho 5U, V).Icy'ingenzi, twabonye ubwiyongere bukabije bwa β-catenine (Igishusho 5W, X) na E-cadherine (Igishusho 5Y, Z) ku mbibi z’imitsi.Twasuzumye kandi β-catenin yandujwe na NCC mu mitsi y’imitsi ya E8.5 (Ishusho 5K, L).irangi-catenin ryasaga nkaho rikomeye muri Specc1l mutant neural folds (Igishusho 5L na K), byerekana ko impinduka za AJ zatangiye.Muri micrografi ya elegitoronike yibice bya gihanga bya E9.5, twongeye kubona ubwiyongere bwa diffuse electron-yuzuye irangi mu nsoro za Specc1l ugereranije na WT (Ishusho 5AA, BB na S1E-H).Dufatiye hamwe, ibisubizo bishyigikira ibisubizo bya vitro muri selile SPECC1L-kd U2OS kandi byerekana ko AJ irangi ryabanjirije icyiciro cya CNCC mungingo zacu za mutant.
Urebye isano izwi yo kurwanya ibikorwa bya AKT hamwe na E-cadherin itajegajega, 17,28 twavuze ko uruhare rwa signal ya PI3K-AKT.Twongeyeho, twabonye ibibyimba bitagaragara muri zimwe mu nsoro zacu za mutant zatorotse urupfu (<5%) kuri E9.5-10.5 hanyuma dutura hafi ya E13.5 (Ishusho S3).Subepidermal vesicles nikimenyetso cyo kugabanya ibimenyetso bya PI3K-AKT bishingiye kuri PDGFRα12.Fantauzzo n'abandi..Mubyukuri, urwego rwa pan-AKT hamwe na fosifori ikora Ser473-AKT yagabanutse muri vivo muri tissue ya mutant ya Specc1l kugeza ifatwa rya E9.5 (ishusho 6A-D).Kugabanuka kurwego rwa fosifora Ser473-AKT birashobora guterwa rwose no kugabanuka kurwego rwa pan-AKT muri vivo (FIG. 6E) no muri vitro (FIG. 6F).Kugabanuka kwa vitro byagaragaye gusa mugihe selile U2OS zahujwe cyane nimpinduka zimiterere ya selile nubucucike bwa AJ (Ishusho 6D).Rero, amakuru yacu yerekana ko SPECC1L ari igitabo gishya cyiza cya PI3K-AKT cyerekana muri morfogenezi ya craniofacial.
. , ugereranije no kugenzura WT.Guhindura iburengerazuba byakorewe ku bwoko bwa lysates na mutant lysates mubihe bimwe.Amashusho yerekanwe kuri SPECC1L yakuwe kumurongo umwe.Blot imwe yakuweho hanyuma yongera gusuzumwa hamwe na anti-pan-ACT na β-actin antibodies.Urwego rwa Pan-AKT mubice bya E8.5 (A, B) nurwego rwa fosifora S473-AKT mubice bya E9.5 byagabanutse cyane..Utubari twibeshya twerekana SEM uhereye kubintu bitatu byigenga byiburengerazuba..Imisoro ya 11 ya mutant yerekana ikwirakwizwa ry'utugingo ngengabuzima (I), ariko umubare w'uturemangingo turimo apoptose wiyongereye cyane (J).
Twahise dusuzuma ibimenyetso byo gukwirakwizwa na apoptose.Ntabwo twabonye itandukaniro riri hagati yo gukwirakwiza insoro za E9.5 (Igishusho 6E, G ugereranije na I) hamwe n’ikwirakwizwa rya 82.5% kuri mutant WT na 86.5% kuri mutant ya Specc1l yapimwe na KI67 yanduye (p <0.56, Fisher's Fisher ikizamini nyacyo).Mu buryo nk'ubwo, ntitwigeze tubona itandukaniro riri hagati ya apoptose yapimwe no kwanduza caspase 3 yacagaguritse mu mitsi ya E8.5 kugeza igihe urusoro rufatiye (ruterekanwa) (ruterekanwa).Ibinyuranye, apoptose yariyongereye cyane mu nsoro zose za E9.5 (ishusho 6F, H na J).Uku kwiyongera muri rusange kwa apoptose guhuza no kugabanya ibimenyetso bya PI3K-AKT no kwica hakiri kare 29,30,31.
Ibikurikira, kugirango twemeze uruhare rwimpamvu ya PI3K-AKT yerekana muri AJ impinduka muri selile yacu ya kd, twahinduye muburyo bwa chimique inzira yo kugenzura na selile ya kd (Ishusho 7A-F).Twakoresheje nk'ikimenyetso cyerekana imiterere y'utugari twahinduye phenotype yagaragaye muri selile SPECC1L-kd, twapimye dukoresheje igipimo cy'uburebure burebure (uburebure) n'uburinganire buhagaritse (ubugari).Ikigereranyo cya 1 giteganijwe kuri selile zingana cyangwa cuboidal (Ishusho 7G).Usibye imiterere y'utugari, twemeje kandi ingaruka kuri AJ ukoresheje β-catenin irangi (Ishusho 7A'-F ').Kubuza inzira ya PI3K-AKT ukoresheje wortmannin byari bihagije kugirango uhindure imiterere ya selile mu ngirabuzimafatizo (Ishusho 7A, C) na AJ (Ishusho 7A ').PI3K-AKT ukora ibikorwa bya SC-79 ntabwo byagize ingaruka kumiterere ya selile (FIG. 7A, E) cyangwa kwaguka kwa AJ (FIG. 7A ') muri selile igenzura.Muri selile SPECC1L-kd, gukomeza guhagarika inzira ya PI3K-AKT byatumye apoptose yiyongera (Igicapo 7B, D) ndetse no kwiyongera kugaragara kwa β-catenine (Igicapo 7B '), bihuye nibyacu muri vivo ihindagurika.Icy'ingenzi, gukora inzira ya PI3K-AKT yazamuye cyane imiterere ya selile (Igicapo 7B, F) na AJ phenotypes (Ishusho 7B ”).Impinduka mu miterere y'utugari zapimwe nk'ikigereranyo cyo kuzenguruka ingirabuzimafatizo (CCR) kandi ugereranije n'akamaro nk'uko byasobanuwe haruguru (FIG. 7G).Mubyukuri, muri selile zigenzura (Igicapo 7G, CCR = 1.56), kuvura wortmannin byari bihagije kugirango uhindure cyane imiterere yimikorere (ishusho 7G, CCR = 3.61, p <2.4 × 10-9) kurwego rusa nubwavuzwe muri SPECC1L.-kd selile (Ishusho 7G, CCR = 3.46).Ubuvuzi bwa Wortmannin bwo kuvura selile SPECC1L-kd (Igicapo 7G, CCR = 3.60, ntarengwa) nticyari gikomeye kuruta ingirabuzimafatizo za kd zitavuwe (Ishusho 7G, CCR = 3.46, zidakwiye) cyangwa ingirabuzimafatizo zivura wortmannin (Ishusho 7G)., CCR = 3.46, birengagije) byongeye bigira ingaruka kumurambararo (7G, CCR = 3.61, birengagije).Icy'ingenzi cyane, umukoresha wa SC-79 AKT yagaruye phenotype ndende ya selile ya SPECC1L-kd (Ishusho 7G, CCR = 1.74, p <6.2 × 10-12).Ibisubizo byemeza ko SPECC1L igenga ibimenyetso bya PI3K-AKT ikanagaragaza ko kugabanuka gukabije kwa SPECC1L bigira ingaruka ku gufatira ku ngirabuzimafatizo, mu gihe igabanuka rikomeye ritera apoptose (Ishusho 8).
. .Utugingo ngengabuzima tutavuwe ni cuboidal (A) hamwe na β-injangwe isanzwe ya selile (A '), mugihe kd selile irambuye (B) hamwe niyongera β-injangwe (B').Nyuma yo guhagarika inzira ya PI3K-AKT, ingirabuzimafatizo zaguye (C) hamwe no kwaguka kwinjangwe (C '), mugihe kd selile zatangiye kwandura apoptose (D), zisa na misoro yacu ihindagurika cyane kandi yerekana β-injangwe yazamutse cyane.irangi (D ').Nyuma yo gukora inzira ya PI3K-AKT, selile zo kugenzura zagumye kuba cuboidal (E) kandi zifite ibisanzwe cat-injangwe (E '), mugihe kd selile yerekanaga neza imiterere yimikorere ya selile (F) na cat-injangwe (F'), byerekana .Utavuwe (NT) SPECC1L-kd selile (CCR = 3.46) yari ndende cyane kuruta selile igenzura (CCR = 1.56, p <6.1 × 10–13).Wort yabujije inzira ya PI3K-AKT mu ngirabuzimafatizo zari zihagije kugira ngo habeho kurasa mu buryo bw'akagari (CCR = 3.61, p <2.4 × 10-9).Muri ubwo buryo, AKT ikora na SC-79 muri selile SPECC1L-kd yagaruye ingirabuzimafatizo kugirango igenzure urwego (CCR = 1.74, p <6.2 × 10–12).Ubuvuzi bwa Wortmannin bwo kuvura selile SPECC1L-kd bwatumye apoptose yiyongera ariko nta kongera kwiyongera kwimiterere yimikorere ya selile (CCR = 3.60) ugereranije na kd itavuwe (CCR = 3.46, ns) cyangwa selile igenzura ya wortmannin (3.61) yagaragaye muri.ns = ntacyo bitwaye.+/- Ibipimo bya SEM kuri selile 50 birerekanwa.Itandukaniro ryibarurishamibare ryabazwe ukoresheje t-test yabanyeshuri.
(A) Igishushanyo mbonera cyo kubuza no gukora inzira ya PI3K-AKT bigatuma AJ ihinduka no gutabara, kimwe.(B) Icyitegererezo cyatanzwe na AKT protein ihindagurika na SPECC1L.
CNCCs yimuka isaba AJ lysis gutandukana nubwonko bwimbere bwimbere ya neuroepithelial selile1,15,32.Kwiyongera kwanduza ibice bya AJ no gutakaza ikwirakwizwa rya apical-basal AJ asimmetricike muri selile zabuze SPECC1L haba muri vitro ndetse no muri vivo, bifatanije no kuba hafi ya SPECC1L na β-catenin, byerekana ko imikorere ya SPECC1L kugirango ibungabunge neza umutekano wa AJ kugirango imitsi yumuteguro.actin cytoskeleton.Ihuriro rya SPECC1L hamwe na actin cytoskeleton na β-catenin hamwe no kwiyongera kwumubare wamafirime ya firimu ya acin mugihe SPECC1L idahari bihuye nubwiyongere bwagaragaye bwubwinshi bwa AJ.Ikindi gishoboka nuko umubare wiyongereye wa fibre ya actin muri selile-SPECC1L ibura biganisha ku mpinduka ziterwa nimpagarara.Kuberako guhangayikishwa na selile bigira ingaruka kuri AJ 33 imbaraga, impinduka za voltage zirashobora gukwirakwira cyane AJ 34.Impinduka zose rero zizagira ingaruka kuri CNCC.
Wnt1 igaragarira mubice byambere byubwonko butanga ingirabuzimafatizo.Rero, Wnt1-cre umurongo ukurikirana ibimenyetso byombi mbere na bimuka NCC35.Ariko, Wnt1 iragaragaza kandi clone ya dorsal yubwonko nayo ikomoka kumitsi ya kare 35,36, bigatuma bishoboka ko kwanduza E9.5 mutant ya marike ya Wnt1 mubitereko bifunguye atari CNCC.Ibyiza byacu kubimenyetso bya NCC AP2A na SOX10 byemeje ko imitsi igaragara ya misoro ya specc11 ya mutant yari irimo CNCC.Byongeye kandi, kubera ko AP2A na SOX10 ari ibimenyetso byerekana kwimuka hakiri kare NCC, irangi ryiza ryerekanaga ko utugingo ngengabuzima CNCC nyuma yo kwimuka idashobora gutondekwa na E9.5.
Amakuru yacu yerekana ko amabwiriza ya molekuline ya AJ na SPECC1L yahujwe na signal ya PI3K-AKT.Ikimenyetso cya AKT kigabanutse muri selile na tissue zabuze SPECC1L.Ibyavuye muri Fantauzzo n'abandi.shyigikira uruhare rutaziguye kuri PI3K-AKT amarenga muri morfogenezi ya craniofacial.(2014) yerekanye ko kubura ibikorwa bya PDGFRα bishingiye kuri PI3K-AKT byerekana ibimenyetso biganisha kuri fenotype.Twerekana kandi ko kubuza inzira ya PI3K-AKT birahagije kugirango uhindure AJ nuburyo bwimikorere muri selile U2OS.Bihuye nibyo twabonye, ​​Kayini n'abandi.37 yerekanye ko kugabanya PI3K α110 subunit muri selile endothelia bituma habaho kwiyongera gutya kwa pericellular β-catenin irangi, byitwa kwiyongera kwa "indangagaciro yo guhuza".Nyamara, muri selile ya endoteliyale ifite filime ya actin yamaze gutegurwa cyane, guhagarika inzira ya PI3K-AKT bivamo imiterere ya selile.Ibinyuranye, selile SPECC1L-kd U2OS yerekanye imiterere ya selile ndende.Itandukaniro rishobora kuba ubwoko bwimikorere yihariye.Mugihe guhagarika ibimenyetso bya PI3K-AKT bigira ingaruka burundu kuri actin cytoskeleton, ingaruka kumiterere ya selile igenwa nimpinduka zimpagarara ziterwa nimpinduka zubucucike no gutunganya fibre nkuru.Muri selile U2OS, twakoresheje gusa imiterere ya selile ihinduka nkikimenyetso cya SPECC1L-idafite AJ ihinduka no gukira.Mu gusoza, turakeka ko kubuza inzira AKT kubura kwa SPECC1L byongera AJ ituze kandi bikagabanya gusohora muri CNCC.
Igishimishije, urwego rwa pan-AKT rwaragabanutse muri vitro no muri vivo usibye urwego rwa fosifori 473-AKT mugihe SPECC1L idahari, byerekana ko hashyirwaho amabwiriza ya PI3K-AKT yerekana urwego rwa AKT ihagaze neza cyangwa igurishwa.Imirasire ya SPECC1L na MID1, byombi bifitanye isano na syndrome ya Opitz / GBBB, ikora poroteyine zihagarika microtubules 18,22.Uburyo SPECC1L na MID1 bahuza microtubule itajegajega ntabwo byumvikana neza.Kubireba SPECC1L, iyi stabilisation ikubiyemo acetylation yongerewe igice cya microtubules 18.Birashoboka ko SPECC1L ikoresha uburyo busa kugirango ihagarike izindi poroteyine nka AKT.Byerekanwe ko acetylation y ibisigazwa bya lysine muri proteine ​​ya AKT itera kugabanuka kwa membrane hamwe na fosifora38.Byongeye kandi, hose kumurongo wa K63 kumurongo umwe wa lysine usigaye kuri AKT urasabwa kugirango membrane ibeho kandi ikore 39,40.Mu bintu byinshi bifitanye isano na poroteyine za SPECC1L zagaragaye mu musemburo mwinshi winjiza umusemburo wa kabiri-bivangavanze, bine - CCDC841, ECM2942, APC na UBE2I43 - byagize uruhare mu guhinduranya poroteyine cyangwa gutuza binyuze ahantu hose cyangwa sumoylation.SPECC1L irashobora kugira uruhare muguhindura nyuma yubusobanuro bwibisigisigi bya AKT lysine, bikagira ingaruka kuri AKT.Icyakora, uruhare rukomeye rwa SPECC1L muguhindura no gutuza kwa poroteyine AKT iracyasobanurwa.
Inenge zikomeye mu mvugo ya SPECC1L muri vivo zatumye ikimenyetso cya AJ cyiyongera kandi cyuzuye CNCC cyuzuye, ndetse no kwiyongera kwa apoptose no kwica hakiri kare.Raporo zabanje zerekanye ko ihinduka ryimbeba nimbeba ziyongereye kurwego rwa apoptose zifitanye isano nindwara zifata imitsi 44,45,46,47 nindwara ya craniofacial48.Byagaragaye ko urupfu rwinshi rwimitsi mu mitsi cyangwa imitsi ya pharyngeal rushobora kuvamo ingirabuzimafatizo zidahagije zikenewe kugirango morfogeneque ikore neza 48,49,50.Ibinyuranye, imirongo ya selile idafite SPECC1L yagabanutse mu buryo bugaragara imvugo ya SPECC1L yerekanaga impinduka za AJ gusa nta kimenyetso cyerekana ko urupfu rwiyongereye.Nyamara, kubuza imiti inzira ya PI3K-AKT muri selile ya Kd byatumye apoptose yiyongera.Rero, kugabanuka kugereranije imvugo cyangwa imikorere ya SPECC1L ituma selile ibaho.Ibi bihuye no kureba ko insoro zidasanzwe za Specc11 zihunga gutabwa muri st.E.Ikindi gihuye nibi ni ibintu bidasanzwe biboneka mu nsoro za heterozygous Specc1l hamwe na craniofacial idasanzwe - birashoboka ko byatewe no gufata neza gene-kimwe no gusanga muri zebrafish aho imwe muri ebyiri za SPECC1L orthologues (specc1lb) zitera fenotipi itinze, harimo no gutakaza intanga ngore; urwasaya rwo hepfo hamwe n'ibice byombi.Rero, heterozygous SPECC1L gutakaza-imikorere-ihinduka ryimikorere yagaragaye mubarwayi babantu irashobora gutera ubumuga buke mumikorere ya SPECC1L mugihe cya morfogenezi ya craniofacial, bihagije kugirango basobanure ibice byabo bya orofacial.SPECC1L ishingiye kumabwiriza yimibonano mpuzabitsina irashobora kandi kugira uruhare muri palatogenezi no guhuza imiyoboro yimitsi.Ubundi bushakashatsi bwakozwe kumikorere ya SPECC1L buzafasha gusobanura uruhare rwumubano wigihe gito muri CNCC mugihe cyo gufunga imitsi ya neuroepithelial selile na craniofacial morphogenez.
Kugenzura U2OS osteosarcoma na selile SPECC1L-kd byasobanuwe mbere (Saadi et al., 2011).Antibodies zirwanya SPECC1L nazo zaranzwe mbere (Saadi et al., 2011).Antibodiyite zirwanya cat-catenin (urukwavu; 1: 1000; Santa Cruz, Dallas, TX) (imbeba; 1: 1000; Ikoranabuhanga ryerekana ibimenyetso, Danvers, MA), myosin IIb (1: 1000; Sigma-Aldrich, St. ), MO)), E-cadherin (1: 1000; Abkam, Cambridge, MA), AP2A (1: 1000; Novus Biologicals, Littleton, Colo.), SOX10 (1: 1000; 1000; Ibinyabuzima bya Aviva Sisitemu, San Diego . ). MO) yakoreshejwe nkuko byasobanuwe..Amashusho ya Actin yandujwe na Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Utugingo ngengabuzima U2OS hamwe na selile SPECC1L-kd byatejwe imbere muri glucose isanzwe DMEM yongerwaho na serumu 10% ya serumu (Life Technologies, Carlsbad, CA).Ku mpinduka za AJ, selile 2 x 105 zatewe ku kirahure zivuwe na 0.1% ya gelatine ya porcine (Sigma-Aldrich, St. Louis, MO) kandi ireba impinduka mu miterere y'utugari.Ingirabuzimafatizo zegeranijwe ku bihe bitandukanye byerekanwe: amasaha 4 nyuma yo gutera (t = 1), amasaha 24 nyuma yo gutera (t = 2), guhuza nta guhinduka kwimiterere ya selile (t = 3), guhindura imiterere ya selile (t = 4) , 24 h nyuma yimiterere yimikorere ya selile (t = 5) na 48 h nyuma yimiterere ya selile (t = 6) (Ishusho 1, 2, 3).Kugirango uhindure inzira ya PI3K-AKT, selile zarezwe mumitekerereze yerekanwe hamwe na PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) cyangwa SC-79 ukora (TOCRIS Biosciences, Minneapolis Adams, Minnesota).Uburyo burimo imiti bwahinduwe buri munsi.
Amajwi yafashwe kumurongo yakozwe mugucunga neza na selile ya KD mubihe bisanzwe byumuco, kandi amashusho atandukanye yo gukusanya yakusanyirijwe buri minota 10 muminsi 7.Amashusho yabonetse hakoreshejwe mudasobwa igenzurwa na mudasobwa Leica DM IRB ihinduranya microscope ifite ibyuma bya mashini hamwe nintego ya 10 × N-PLAN ihujwe na kamera ya QImaging Retiga-SRV.Mu gihe cyo gufata amashusho, imico y'utugari yagumishijwe kuri 37 ° C mu kirere cyuzuye na 5% CO2.
Imirongo ibiri ya gene ya ES selile DTM096 na RRH048 yo mukarere ka Mutant Mouse Resource Centre (UC Davis, CA) yakoreshejwe kugirango habeho imirongo yimbeba yabuze Specc11, yagenwe Specc1lgtDTM096 na Specc1lgtRRH046.Muri make, selile 129 / REJ ES zatewe muri C57BL6 blastocysts.Imbeba za chimeric zavuyemo zororerwa hamwe nimbeba C57BL6 zumugore kugirango hamenyekane urubyaro rufite ikoti rya agouti.Kubaho gene trap vector yinjizwamo yakoreshejwe kugirango tumenye heterozygote.Imbeba zabitswe inyuma ivanze ya 129 / REJ; C57BL6.Ahantu hashyizwemo urubuga rwumutego wa genetique byemejwe na RT-PCR, uko genome ikurikirana, hamwe no kuzuzanya kwa geneti (Ishusho yinyongera 1).Kugirango ukurikirane umurongo wa CNCC wimbeba ebyiri za heterozygous Specc1lGT, imbeba za ROSAmTmG (# 007576) na Wnt1-Cre (# 003829) (Laboratoire ya Jackson, Bar Harbour, ME) zambutse kugirango zibyare insoro za ROSAmTmG na Wnt1-Cre allele.Ubushakashatsi bwose bwakozwe ku mbeba bwakozwe hakurikijwe protocole yemejwe na komite ishinzwe kwita ku nyamaswa n’imikoreshereze y’ikigo cy’ubuvuzi cya kaminuza ya Kansas.
Imbuto zashyizwe muri (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) muminota 60 mubushyuhe bwicyumba.Nyuma yo gukosorwa muri X-gal irangi (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, mM 2 MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg / ml X-gal) Iterambere ryakozwe ryakozwe kuri 37 ° C .° C mu masaha 1-6.Embryos zashyizwe nyuma muri 4% PFA kandi igaragara.
Kubijyanye no guhindagura iburengerazuba, selile zatewe muri buffer ya lysis pasiporo (Promega, Fitchburg, WI) hiyongeraho imvange ya HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO).Lysates yatunganijwe kuri 12% polyacrylamide Mini-PROTEAN TGX geles yiteguye (Bio-Rad, Hercules, CA) hanyuma yimurirwa muri Immobilon PVDF membrane (EMD Millipore, Billerica, MA).Ibibyimba byafunzwe mumata 5% muri PBS arimo 0.1% Hagati.Antibodies yashizwemo ijoro ryose kuri 4 ° C cyangwa isaha imwe mubushyuhe bwicyumba.Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) yakoreshejwe mugutanga ibimenyetso.Kubikingira, insoro zashizweho ijoro ryose muri 4% PFA / PBS hamwe na cryopreserved.Tissue cryosections yahagaritswe muri PBS irimo serumu yihene isanzwe 1% (Thermo Scientific, Waltham, MA) na 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) hanyuma yinjizwa kuri 4 ° C muri incubator mugihe cya ijoro.hamwe na antibody irwanya antibody na fluorescent ya kabiri (1: 1000) kumasaha 1 kuri 4 ° C.Ibice bisize byashyizwe muri zahabu ya ProLong (Thermo Scientific, Waltham MA) kandi amashusho meza yabonetse hakoreshejwe microscope ya Leica TCS SPE.Buri immunostaring yakozwe nkubushakashatsi butatu bwigenga kuri cirossection byibura insoro ebyiri za mutant.Ikigereranyo gihagarariye kirerekanwa.
Ingirabuzimafatizo zashyizwe muri buffer ya RIPA yahinduwe (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, na HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) Muri make, lysates yabanjirijwe hamwe na protein G magnetique (Life Technologies, Carlsbad, CA) hanyuma yinjizwa ijoro ryose kuri 4 ° C. hamwe na anti-SPECC1L cyangwa IgG proteine ​​G ya proteine ​​G yakoreshejwe mugukuramo SPECC1L hanyuma blotting yiburengerazuba ikorwa hakoreshejwe anti -β-catenin antibody yasobanuwe haruguru Ubushakashatsi bwa co-IP bwerekanwe bugereranya ubushakashatsi bune bwigenga.
Utugingo ngengabuzima tumwe na tumwe cyangwa intangangore z'imbeba zahawe ikigo cya microscopi electron kuri santere ya kaminuza ya Kansas.Muri make, ibyitegererezo byashyizwe muri resin ya EMbed 812 (Electron Microscopy Science, Fort Washington, PA), polymerized ijoro ryose kuri 60 ° C, hanyuma igabanywa kuri 80 nm ikoresheje ultramicrotome ya Leica UC7 ifite icyuma cya diyama.Ibice byerekanwe hakoreshejwe JEOL JEM-1400 yohereza electron microscope ifite imbunda ya kV 100 ya Lab6.
Uburyo bwo gutanga iyi ngingo: Wilson, NR n'abandi.Kubura SPECC1L biganisha ku kongera ituze ryingingo zifatanije no kugabanya gusibanganya ingirabuzimafatizo ya cranial neural crest selile.siyanse.6, 17735;doi: 10.1038 / srep17735 (2016).
Uwera-Jeanne, J.-P.Kwinjiza no gutandukanya imitsi.(Ubumenyi bwa Springer + Itangazamakuru ryubucuruzi; Landes Bioscience / Eurekah.com, 2006).
Cordero, DR n'abandi.Cranial neural crest selile igenda: uruhare rwabo mugutezimbere craniofacial.Ikinyamakuru cyo muri Amerika cyubuvuzi.Igice A 155A, 270-2279, doi: 10.1002 / ajmg.a.33702 (2011).
Boland, RP Neurocristopathia: gukura kwayo niterambere mumyaka 20.Umuganga w'abana.indwara.laboratoire.ubuvuzi.17, 1-25 (1997).
Mangold E., Ludwig KU na Noten MM Iterambere muri genetics ya orofacial clefts.Imigendekere yubuvuzi bwa molekile 17, 725–733, doi: 10.1016 / j.molmed.2011.07.007 (2011).
Minu, M. na Riley, FM Uburyo bwimikorere ya cranial neural crest selile yimuka no gushushanya mugihe cyiterambere rya craniofacial.Iterambere 137, 2605-22621, doi: 10.1242 / dev.040048 (2010).
Dixon, MJ, Marazita, ML, Beaty, TH na Murray, JK Cleft iminwa n'amagage: gusobanukirwa n'ingaruka z'ibidukikije n'ibidukikije.ibisobanuro bisanzwe.Intangiriro 12, 167–178, doi: 10.1038 / nrg2933 (2011).
Ingram, CR n'abandi.Imiterere idasanzwe y'uruhu, ingingo, n'akarere ka craniofacial mubintu bigenga interferon-6 (Irf6) -imbeba zidafite akamaro.Genette y'igihugu.38, 1335–1340, doi: 10.1038 / ng1903 (2006).
Peyrard-Janvid, M. n'abandi.Guhinduka kwinshi muri GRHL3 bitera syndrome ya van der Waord no kubangamira iterambere rya periderm yo mu kanwa.Am J Hum Genet 94, 23–32, doi: 10.1016 / j.ajhg.2013.11.009 (2014).
Harris, MJ na Juriloff, DM Kuvugurura urutonde rwimiterere yimbeba zifite inenge zo gufunga imiyoboro yimitsi no gutera imbere biganisha kumyumvire yuzuye ya genetike yo gufunga imiyoboro.Iperereza ku nenge zavutse.Igice A, Ubuvuzi bwa Clinical na Molecular Teratologiya 88, 653–669, doi: 10.1002 / bdra.20676 (2010).
Fantauzzo, KA & Soriano, P. PI3K yunganirwa na PDGFRalpha yerekana kugenga kubaho no gukwirakwira mu mikurire ya skeletale binyuze mu nzira ya p53 ishingiye ku mitsi.Iterambere rya Gene 28, 1005–1017, doi: 10.1101 / gad.238709.114 (2014).
Kopp, AJ, Icyatsi, ND na Murdoch, JN Disheveled: Isano yo kwaguka guhuza no gufunga imitsi.Inzira muri neurologiya.26, 453–455, doi: 10.1016 / S0166-2236 (03) 00212-1 (2003).