ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube Don Abubuwan Sinadarai na Masana'antar Sinadaran, Rashin ƙarancin SPECC1L yana haifar da haɓakar kwanciyar hankali na haɗin gwiwa da rage zubar da ƙwayoyin crest cranial.

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ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube For Chemical Industry

Liaocheng Sihe SS Material Co., Ltd.babban masana'anta ne wanda ya kware a cikin bututun bakin karfe, bututu masu haske, bututu mai nade da sauransu.Domin sauƙaƙa abokan ciniki, muna da bututu da welded kuma.Liaocheng Sihe SS Material Co., Ltd.yana da kayan aikin samarwa da gwaji mafi inganci.Za mu iya cika bukatunku gaba ɗaya.Dangane da ƙayyadaddun ƙayyadaddun ƙayyadaddun ƙayyadaddun bututu waɗanda muke samarwa koyaushe suna da daidaitaccen juriyar OD da WT.Ikon haƙuri yana daidai da samar da ƙa'idodi.Samfuranmu koyaushe suna gamsu da abokan ciniki.Abokan ciniki sun sayi samfuran mu sun haifar da ƙarin riba.
a) OD (Diamita na waje): 3.18mm zuwa 101.6mm
b) WT (Kaurin bango): 0.5mm zuwa 20mm
c) Length: Bisa ga abokin ciniki ta bukata
d) Matsayi: ASTM A312;ASTM A269;ASTM A789;ASTM A790 da dai sauransu
e) Hanyar tsari: ERW, EFW da dai sauransu

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Kwayoyin jijiyoyi na cranial (CNCC) sun yi nisa daga ɓangarorin ƙwararrun amfrayo kuma suyi ƙaura zuwa gaɓar ɓangarorin pharyngeal, waɗanda ke samar da mafi yawan sifofin tsakiya.Rashin aikin CNCC yana taka muhimmiyar rawa a cikin ilimin etiology na ƙwanƙwasa orofacial, rashin lafiya na gama gari.An sami maye gurbi na Heterozygous SPECC1L a cikin marasa lafiya da ke da ɓangarorin ɓangarorin ɗabi'a.Anan, mun bayar da rahoton ingantaccen tabo na abubuwan haɗin gwiwar mannewa (AJ), β-catenin da E-cadherin a cikin ƙwayoyin ƙwanƙwasawa na SPECC1L, da micrographs na lantarki suna nuna yaduwar apical-basal na AJ.Don fahimtar rawar SPECC1L a cikin morphogenesis na craniofacial, mun ƙirƙiri ƙirar linzamin kwamfuta mai ƙarancin Specc1l.Mutantan Homozygous suna da kisa na amfrayo kuma suna nuna raunin ƙulli bututun jijiya da lamination na CNCC.Ana ƙara tabon furotin AJ a cikin folds na jijiyoyi.Wannan lahani na AJ yayi daidai da lahani a cikin CNCC delamination, yana buƙatar rushewar AJ.Bugu da kari, Specc11 mutants sun rage siginar PI3K-AKT da haɓaka apoptosis.A cikin vitro, ƙarancin hana siginar PI3K-AKT a cikin nau'in nau'in daji ya isa ya haifar da canje-canjen AJ.Mahimmanci, canje-canjen AJ da SPECC1L ke haifarwa ana iya juyawa ta kunna hanyar PI3K-AKT.A hade, waɗannan bayanan suna ba da shawarar cewa SPECC1L, a matsayin sabon mai tsara siginar PI3K-AKT da ilimin halitta AJ, ana buƙatar rufe bututun jijiya da CNCC stratification.
Cranial neural crest Kwayoyin (CNCCs) sun daidaita zuwa dorsal neuroectoderm kuma sun rabu da neuroepithelium na jijiyar jijiyoyi masu tasowa ta hanyar tsarin da ya shafi canjin epithelial-mesenchymal (EMT) 1,2,3.Premigrating epithelial CNCCs sun rushe tsaka-tsakin tsaka-tsakin salula kuma sun zama CNCC masu ƙaura na mesenchymal waɗanda ke cike arches na farko da na biyu na pharyngeal kuma suna samar da mafi yawan gungumen craniofacial.Don haka, kwayoyin halittar da ke daidaita ayyukan CNCC galibi suna rushewa a cikin ilimin ilimin cututtukan cututtukan cututtukan craniofacial kamar clefts na orofacial, galibi suna shafar haihuwar 1/800 a cikin Amurka kaɗai.Daya daga cikin nakasar haihuwa8.
Delamination na CNCC ya zo daidai da rufe bututu na gaba tsakanin 8.5 zuwa 9.5 kwanakin ci gaban amfrayo a cikin mice.Mutants na adadin ƙwayoyin linzamin kwamfuta masu alaƙa na orofacial suma suna nuna wani nau'i na lahani na bututun jijiya, gami da Irf69,10, Ghrl310, Cfl111, da Pdgfrα12.Koyaya, ana iya ɗaukar matakan rufe bututun jijiya da CNCC stratification masu zaman kansu, kamar yadda Slotch mutant linzamin kwamfuta (Pax3) yana nuna lahani a cikin rufewar bututun jijiyoyi ba tare da wani tasiri akan CNCC stratification ko ƙaura 13,14 ba.Ƙarin ƙirar linzamin kwamfuta tare da lahani a cikin rarrabawar CNCC da kuma rufe bututun jijiyoyi zai taimaka ƙaddamar da tushen kwayoyin gama gari na waɗannan matakai guda biyu.
Warewa CNCC daga ƙwayoyin neuroepithelial yana buƙatar rushewar haɗin gwiwa (AJs), waɗanda suka ƙunshi hadaddun furotin da suka ƙunshi, da sauransu, E-cadherin, β-catenin, α-E-catenin, da α-actinin da ke hade da actin filaments 2. Nazarin wuce gona da iri E-cadherin a cikin folds na jijiyoyi ya nuna raguwa ko jinkiri a cikin CNCC delamination.Sabanin haka, ƙaddamar da E-cadherin yana haifar da ƙaddamarwa na farko15,16.Yawancin abubuwan da ke daidaita EMT yayin ƙaddamarwar CNCC sune abubuwan rubutun (AP2a, Id2, FOXD3, SNAIL, TWIST, SOX10) da kuma matrix na extracellular (ECM) sunadaran gyaran gyare-gyare kamar matrix metalloproteinases (MMPs), duk da haka CNCCs sune masu sarrafa cytoskeletal AJ kai tsaye. har yanzu ba a sani ba.Hanyar PI3K-AKT sananne ne don adawa da matakan E-cadherin, galibi daga binciken kansa17.Nazarin kwanan nan sun nuna cewa asarar siginar PI3K-AKT na tushen PDGFa a cikin mice yana haifar da rashin daidaituwa na craniofacial, gami da ƙwanƙwasa ƙwanƙwasa da lahanin bututun jijiya12.Koyaya, alaƙar da ke tsakanin hanyar PI3K-AKT da kwanciyar hankali na AJ akan madaidaicin CNCC ba ta da tabbas.
A baya mun gano SPECC1L a matsayin kwayar halitta ta farko a cikin mutane biyu tare da tsaga mai tsanani wanda ya tashi daga baki zuwa ido, wanda aka sani da oblique cleft (ObFC) ko Tessier IV18 cleft.An gano maye gurbi na SPECC1L a cikin iyalai guda biyu masu yawa tare da ciwon Opitz G/BBB wanda ya mamaye kansa (OMIM #145410), wanda mutanen da abin ya shafa suka nuna hyperdistance da cleft lebe / palate19, kuma a cikin iyali guda tare da ciwon Tibi overdistance syndrome (OMIM #145420)20 .fiye da rabin lokuta na ciwon Opitz G/BBB suna da alaƙa X (OMIM #300000) kuma ana haifar da su ta hanyar maye gurbi a cikin kwayar halitta ta MID1, wanda ke ɓoye sunadaran 22 na kwarangwal mai alaƙa da microtubule.Muna tsammanin cewa SPECC1L, kuma sunadaran da ke hade da microtubules da actin cytoskeleton, na iya yin sulhu da siginar da ake buƙata don gyaran gyare-gyaren cytoskeleton na actin a lokacin haɗin sel da ƙaura 18.Ta hanyar nazarin in vitro da in vivo, yanzu mun bayyana SPECC1L a matsayin sabon mai tsara zaman lafiyar AJ ta hanyar siginar PI3K-AKT.A matakin salon salula, ƙarancin SPECC1L ya haifar da raguwa a matakin furotin pan-AKT da karuwa a cikin watsawar apical-basal na AJ, wanda aka kawar da shi ta hanyar kunna sinadarai na hanyar AKT.A cikin vivo, embryos masu ƙarancin Specc11 suna nuna ƙarancin rufe bututun jijiya da rage rarrabawar CNCC.Don haka, SPECC1L yana aiki a cikin ingantaccen siginar tushen mannewa tantanin halitta da ake buƙata don aikin CNCC na yau da kullun yayin morphogenesis na fuska.
Don siffanta rawar SPECC1L a matakin salon salula, mun yi amfani da ƙarancin layin osteosarcoma cell U2OS da aka kwatanta a baya a cikin SPECC1L18.Wadannan barga U2OS Kwayoyin tare da SPECC1L (kd) knockdown suna da matsakaici (60-70%) ragewa a cikin matakan SPECC1L kwafi da sunadarai, tare da lahani a cikin ƙaura da sake tsarawa na actin cytoskeleton 18. Sabanin haka, raguwa mai tsanani na wucin gadi. An nuna SPECC1L don haifar da lahani na mitotic 23.Bayan ƙarin haɓakawa, mun gano cewa ƙwayoyinmu SPECC1L-kd barga sun canza ilimin halittar jiki a babban matakin haɗuwa (Hoto 1).Kwayoyin kulawa ɗaya ɗaya da ƙwayoyin kd a ƙananan haɗuwa sunyi kama (Hoto 1A, D).Sa'o'i 24 bayan haɗuwa, sel masu sarrafawa sun riƙe siffar siffar su (Fig. 1B, E), yayin da SPECC1L-kd sel elongated (Fig. 1C, F).Girman wannan canji a siffar tantanin halitta an kama shi ta cikin hoton rayuwa mai rai na ƙwayoyin sarrafawa da ƙwayoyin kd (fim 1).Don sanin rawar SPECC1L a cikin sel masu haɗaka, mun fara bincika bayanin sa.Mun gano cewa matakan furotin SPECC1L sun karu akan fusion (Hoto 1G), yayin da matakan kwafin SPECC1L bai karu ba (Hoto 1H).Bugu da ƙari, yayin da ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta ta SPECC1L ta tara a kan iyakoki na tsakiya (Fig. 2A-E), tare da tsarin da ke hade da na β-catenin da ke hade da membrane (Fig. 2A'-E').Ganin haɗin SPECC1L tare da actin cytoskeleton 18,23 mun yi hasashe cewa SPECC1L yana hulɗa tare da haɗin gwiwar mannewa na tushen actin (AJ).
(AF) SPECC1L knockdown (DF) Kwayoyin suna tsawo a babban haɗuwa (F) idan aka kwatanta da sarrafa U2OS Kwayoyin (AC).An nuna a nan uku daga cikin maki shida na lokaci (T1, T3, T6) waɗanda muka zaɓa don ƙananan ƙwayoyin sel daban-daban.(G) Binciken ɓangarorin Yamma yana nuna cewa furotin SPECC1L yana daidaitawa a babban matakin haɗuwa idan aka kwatanta da ƙaramin matakin haɗuwa a cikin ƙwayoyin sarrafawa.Yankin Yamma na SPECC1L yana nuna ƙungiyar 120 kDa da ake tsammanin da kuma maɗaurin nauyin kwayoyin halitta, mai yiyuwa an canza bayan fassarar (*).An gudanar da bincike na ɓangarorin Yamma a ƙarƙashin yanayi iri ɗaya don ƙanƙanta da babban haɗuwa.Hotunan da ke nuna SPECC1L a ƙanana da babban haɗuwa an ɗauko su daga ɓarna iri ɗaya.An cire tabo iri ɗaya kuma an sake gwada shi tare da antibody β-actin.(H) Ƙididdigar RT-PCR mai ƙididdigewa ya nuna babu wani gagarumin canje-canje a matakan rubutun SPECC1L.Sandunan kuskure suna wakiltar SEMs daga gwaje-gwaje masu zaman kansu guda huɗu.
(AE) Mun zaɓi maki shida na lokaci (T1-T6) wanda ke wakiltar kewayon nau'ikan ƙwayoyin sel don daidaita nazarin sigar tantanin halitta da canje-canjen AJ a cikin ƙwayoyin U2OS tare da bugun SPECC1L (kd).Biyar na farko na waɗannan maki lokaci sun haɗa da sel guda ɗaya (T1), 50-70% fusion na ƙananan gungu na tantanin halitta (T2), fusion ba tare da sake fasalin kd Kwayoyin (T3), sake fasalin kd Kwayoyin (T4), da 24 hours canje-canje.a cikin sigar baya na sel kd (T5).An rarraba furotin na SPECC1L mafi yawa a cikin cytoplasm a T1 (A), amma an lura da tarin sa a iyakokin intercellular a wuraren lokaci na gaba (B–E, kibiyoyi).(FJ) β-catenin yana nuna irin wannan tarawa a iyakokin intercellular da ke hade da hadaddun AJ.(A'-E') SPECC1L da β-catenin suna nuna tabo mai rufi a kan iyakokin tantanin halitta a babban yawan tantanin halitta (kibiyoyi).(F'-J') A cikin sel SPECC1L-kd, β-catenin tabo ya bayyana a al'ada a ƙananan ƙwayar sel (F'-H'), amma yana faɗaɗa yayin da siffar tantanin halitta ta canza (I', J'; kibiyoyi), yana nuna cewa AJ sun canza.Bars = 10 µm.
Daga nan mun yi ƙoƙarin tantance tasirin rashi SPECC1L akan AJ.Mun yi amfani da alamomi masu alaƙa da AJ da yawa, gami da abubuwan canonical F-actin, myosin IIb, β-catenin, da E-cadherin24,25,26,27.Actin danniya fibers ya karu a cikin kwayoyin SPECC1L-kd kamar yadda aka bayyana a baya (Fig. 3A, B) 18.Myosin IIb hade da actin filaments ya nuna irin wannan karuwa a cikin kwayoyin SPECC1L-kd a cikin vitro (Fig. 3C, D).β-catenin da ke da alaƙa da AJ yana ɗaure zuwa cadherin a cikin ƙwayar sel, yana nuna alamar magana ta al'ada "zuma" a cikin cubocytes masu sarrafawa (Fig. 3E, G).Abin sha'awa, a cikin hotuna masu lebur ta amfani da microscopy confocal, β-catenin (Fig. 3E,F) da kuma E-cadherin (Fig. 3G, H) da aka lalata a kan kwayar halitta na ƙwayoyin cuta na SPECC1L-rashin sel sun nuna alamun alamun tsawaitawa.Wannan fadadawar β-catenin mai alaƙa da AJ a cikin ƙwayoyin kd ya fi bayyana a haɗuwa, amma ya bayyana ya riga ya canza canje-canje a cikin siffar tantanin halitta (Fig. 2F-J, F'-J').Don tantance yanayin jiki na wannan tsawaita tabon AJ, mun yi nazarin iyakokin tantanin halitta akan saman apical-basal na sel SPECC1L-kd U2OS ta hanyar watsa microscopy na lantarki (TEM) (Hoto 3I, J).Ya bambanta da sel masu sarrafawa (Fig. 3I), wanda ke da yankuna masu yawa na lantarki daban-daban masu nuna alamar AJ (kibiyoyi), kd Kwayoyin (Fig. 3J) sun nuna manyan yankuna masu mahimmanci na babban nau'in lantarki mai nuna alamar AJ tare da jirgin apicobasal..Bugu da ƙari, a kan sassan masu jujjuya, mun lura da ɓangarorin ƙwayoyin sel masu yawa a cikin ƙwayoyin kd (Fig. S1A, B), wanda ke bayyana tsararren ƙirar β-catenin da E-cadherin staining bands (Fig. 3F, H).Don tallafawa rawar SPECC1L a cikin AJs, β-catenin an haɗa shi tare da SPECC1L a cikin lysates na ƙwayoyin U2OS masu haɗuwa (Fig. 3K).Tare da tsawaita rigakafin rigakafi don alamomin AJ, bincike na TEM ya yi daidai da hasashen mu cewa rashi SPECC1L yana ƙaruwa AJ apical-basal yawa da bambance-bambance.
(AH) Ƙara F-actin tabo a cikin sel kd a 48 hours post-fusion (T6; A, B).Canjin tabo na myosin IIb mai alaƙa da F-actin (C, D).Tsarin santsi na β-catenin da E-cadherin membrane tabo a cikin sel masu sarrafawa (E, G) an haɓaka su a cikin ƙwayoyin SPECC1L-kd (F, H).Bars = 10 µm.(I-J) Micrographs na lantarki da ke lura da haɗin gwiwar apical-basal intercellular.Kwayoyin sarrafawa suna nuna yankuna daban-daban masu yawa na lantarki waɗanda ke nuna madaidaicin madaidaicin (I, kibiyoyi).Sabanin haka, gaba dayan mahadar apical-basal a cikin sel SPECC1L-kd sun fito da yawa na lantarki (J, kibiyoyi), suna nuni da ɗimbin yawa da tarwatsa mahaɗar mannewa.(K) β-catenin an haɗa shi tare da SPECC1L a cikin lysates cell U2OS.Hoton da aka ɗauka daga wuri ɗaya yana wakiltar ɗaya daga cikin gwaje-gwaje masu zaman kansu guda huɗu.
Don fahimtar rawar SPECC1L a cikin ƙwayoyin cuta na craniofacial, mun ƙirƙiri ƙirar ƙirar linzamin kwamfuta mai ƙarancin Specc1l ta amfani da layin salula na tarkon ES guda biyu masu zaman kansu, DTM096 da RRH048 (BayGenomics, CA), waɗanda ke wakiltar intron 1 da Specc1l kwafin an kama su a 15 (Fig. 1) .4A, siffa S2).Matsayin genomic na abin da aka saka vector decoy an ƙaddara ta gabaɗayan jerin kwayoyin halitta kuma an tabbatar da shi ta PCR (Fig. S2).Dukansu ƙirar tarkon kwayoyin halitta kuma sun ba da izinin haɗakar da masu ba da rahoto na Specc11-lacZ yayin kamawa.Sabili da haka, an yi amfani da kalmar lacZ da aka ƙaddara ta hanyar X-gal tabon azaman mai nuna alamar Specc11.Dukansu alleles sun nuna irin wannan salon magana na lacZ, tare da tarkon gene DTM096 a cikin intron 1 yana nuna ƙarfi fiye da RRH048 a cikin intron 15 (ba a nuna ba).Koyaya, Specc1l yana bayyana ko'ina, tare da magana mai ƙarfi musamman a cikin ɓangarorin jijiyoyi a E8.5 (Hoto 4B), a cikin bututun jijiyoyi da tsarin fuska a E9.5 da E10.5 (Hoto 4C, D), da kuma haɓaka gaɓoɓi. ku E10.5 da idanu (Hoto na 4D).A baya mun bayar da rahoton cewa SPECC1L magana a cikin farkon pharyngeal baka a E10.5 ya kasance a cikin epithelium da ƙananan mesenchyme18, daidai da layin CNCC.Don gwada maganganun SPECC1L a cikin CNCC, mun yi E8.5 folds jijiyoyi (Figure 4E-J) da E9.5 sassan kwanyar (Hoto 4K-).A E8.5, SPECC1L tabo jijiyar jijiyoyi mai tsanani (Fig. 4E, H), ciki har da sel da aka lalata da alamun NCC (Fig. 4G, J).A E9.5, SPECC1L (Fig. 4K, N) mai ƙarfi mai ƙaura mai ƙaura CNCC tare da AP2A (Fig. 4L, M) ko SOX10 (Fig. 4O, P).
(A) Tsarin tsari na ƙirar ƙirar Specc11 na linzamin kwamfuta yana nuna shigar da lalata vector a cikin ES DTM096 (intron 1) da RRH048 (intron 15) clones cell.(BD) lacZ tabo na heterozygous Specc1lDTM096 amfrayo masu wakiltar Specc1l magana daga E8.5 zuwa E10.5.NE = neuroectoderm, NF = nau'in jijiyoyi, PA1 = farkon pharyngeal baka.(EP) SPECC1L rigakafi tare da alamun NCC AP2A da SOX10 a cikin E8.5 (NF; EJ) folds na jijiyoyi da sassan kwanyar E9.5 (KP).SPECC1L tabo an lura da shi sosai a cikin folds na jijiyoyi E8.5 (E, H; arrowheads), ciki har da sel masu lakabi tare da AP2A (F, G; kiban kiban) da SOX10 (I, J; kibiya).A E9.5, SPECC1L mai tsananin ƙaura CNCCs (K, N; kibiyoyi) masu lakabin AP2A (L, M; kibiyoyi) da SOX10 (O, P; kibiyoyi).
Ketare tsakanin heterozygous Specc1lDTM096/+ da Specc1lRRH048/+ mice yana nuna cewa nau'ikan tarkon kwayoyin halitta guda biyu ba su dace ba kuma wannan fili na heterozygotes da homozygotes na amfrayo na ko dai kwayar tarkon kwayar halitta suna mutuwa ne (Table S1).Matsakaicin Mendelian sun nuna raguwa a cikin adadin rayuwa na heterozygotes a lokacin haihuwa (wanda ake tsammanin 1.34 vs. 2.0).Mun lura da ƙarancin mace-mace a tsakanin heterozygotes, wasu suna da rashin ƙarfi na craniofacial (Fig. S3).Duk da haka, ƙarancin shigar waɗannan dabi'un craniofacial phenotypes yana da wahala a yi nazarin mahimman hanyoyin su na pathophysiological.Don haka, mun mai da hankali kan yanayin halittar ɗan adam na ɗan adam Specc11.
Yawancin mahallin heterozygous ko homozygous Specc1lDTM096 / RRH048 mutant embryos ba su ci gaba ba bayan E9.5-10.5 (Figs. 5A-D), kuma jijiyar jijiyar ba ta rufe gaba (Figs. 5B, D) kuma wani lokacin rufewa a baya (ba a nuna ba). ..Wannan lahani na rufe bututun jijiyoyi yana da alaƙa da yawancin CNCC mai alamar DLX2 da suka rage a cikin folds na jijiyoyi a E10.5, yana nuna babu rarraba (Hoto 5A'-D').Don tantance idan an rage girman girman CNCC shima, mun yiwa layin CNCC alama tare da GFP a cikin layin tarko na mu tare da Wnt1-Cre da ROSAmTmG.Mun jera GFP+ NCC da GFP- (RFP+) wadanda ba NCC ba daga dukan ƴaƴan ƴan mata.A E9.5, rabon CNCC mai alamar GFP mai gudana bai canza sosai tsakanin WT da embryos na mutant (ba a nuna ba), yana nuna ƙayyadaddun CNCC na yau da kullun.Saboda haka, mun yi hasashen cewa ragowar Wnt1-Cre da DLX2 tabo a cikin ɓoyayyen ɓoyayyun jijiyoyi (Hoto 5B') ya kasance saboda rashin lahani na CNCC mai lahani, maiyuwa saboda ƙãra yawa ko tarwatsa ƙwayoyin AJ, kamar yadda aka gani a cikin sel SPECC1L-kd.Mun yi amfani da alamun NCC SOX10, AP2A, da DLX2 don tabbatar da kasancewar CNCC a cikin nau'in jijiya (Hoto 5E-R).A E8.5, an lura da tabon jijiya ga dukkan alamomin NCC guda uku a sassan WT (Fig. 5E, G, I) da Specc1l mutant (Fig. 5F, H, J).A E9.5, yayin da NCC alamomin ƙaura NCC a cikin sassan WT (Fig. 5M, O, Q), an lura da tabon NCC a cikin fallasa jijiyoyi na Specc1l mutant embryos (Fig. 5N, P, R).Saboda SOX10 da DLX2 suna alamar ƙaura CNCCs, wannan sakamakon yana nuna cewa SPECC1L-rashin CNCCs sun cimma ƙayyadaddun ƙayyadaddun ƙaura amma sun kasa yin ƙaura daga ruɓaɓɓen jijiyoyi.
Rancin Specc11 yana haifar da ƙarancin rufe bututun jijiya, lalata ƙwayoyin crest jijiyoyi da AJs.
(A, B') E9.5 WT (A) Embryo ɗauke da ƙaura na cranial crest sel (CNCC) mai lakabi da Wnt1-Cre (A').Sabanin haka, Specc11 mutant embryos suna nuna buɗaɗɗen jijiya (B), kibiya) da CNCC waɗanda basu yi ƙaura ba (B', kibiya).(C, D') Hotunan fili mai haske (C, D') da immunostaining (C', D') na alamar CNCC DLX2 na E10.5 WT embryos (C, C') da Specc1l (D, D').A cikin embryos na WT E10.5, DLX2-tabbatacce CNCC sun mallaki gill arches (C', kibiyoyi), yayin da a cikin mutants, tabo mai ma'ana yana ci gaba da kasancewa a cikin muryoyin jijiyoyi masu buɗewa (D', kibiyoyi) da kuma a cikin rukunan pharyngeal na farko (D', kibiyoyi).) tare da wasu tabo (kibiyoyi) suna nuna rashin ƙarfi da ƙaura na CNCC.ER) Sassan WT da Specc1l mutant embryos a matakan E8.5 (E-L) da E9.5 (M–R) an yi musu lakabi da alamun NCC SOX10 (E, F, M, N), AP2A (G, H, O, P) da DLX2 (I, J, Q, R).A E8.5, an lura da tabon NCC a cikin nau'in nau'in jijiyar daji (NF) da sassan mutant.Haɗin kai na SOX10 da β-catenin a cikin E8.5 WT (K) da mutant (L) sun nuna ƙarar β-catenin tabo a kan iyakokin tantanin halitta a cikin folds na jijiyoyi.A E9.5, an lura da nau'in nau'in daji na CNCCs (M, O, Q) masu ƙaura, yayin da a cikin mutants, CNCC da ba a san su ba sun ba da launi na jijiyoyi (N, P, R).(S-Z) A cikin nazarin alamar alamar vivo AJ a cikin sassan coronal na WT da Specc11DTM096/RRH048 embryos tare da maye gurbin E9.5.Ana nuna kimanin jirgin sama na sashe a kusurwar dama ta sama.A cikin sassan jikin kyallen jikin mutant, an sami ƙarin tabo na F-actin (S, T) da myosin IIb (U, V).Hakazalika da sakamakon in vitro a cikin siffa 3, a cikin embryos masu maye gurbin, an lura da ƙaƙƙarfan launi na membrane don β-catenin (W, X) da E-cadherin (Y, Z).(AA-BB) Micrograph na lantarki na wani sashe na amfrayo mai nau'in daji da ke kallon bayan iyakar tantanin halitta apical-basal yana nuna wani yanki mai yawa na lantarki wanda ke nuni da mannen junctions (AA, kibiyoyi).Sabanin haka, a cikin sassan Specc11 mutant embryos (BB, kibiyoyi), gabaɗayan mahaɗin apicobasal yana da yawa na lantarki, yana nuna ƙarar yawa da tarwatsa mahaɗar mannewa.
Don gwada hasashen mu cewa raguwar yadudduka ya faru ne saboda canjin AJ, mun bincika alamar AJ a cikin fallewar jijiyoyi na Specc1l mutant embryos (Fig. 5S-Z).Mun lura da karuwa a cikin filayen danniya na actin (Fig. 5S, T) da kuma ƙaddamar da ƙaddamarwa na myosin IIB tabo akan filaye na actin (Fig. 5U, V).Mahimmanci, mun lura da ƙarar tabo na β-catenin (Fig. 5W, X) da E-cadherin (Fig. 5Y, Z) a cikin iyakokin intercellular.Mun kuma bincika β-catenin tabo na NCC a cikin jijiyoyi na E8.5 embryos (Fig. 5K, L).β-catenin tabo ya bayyana ya fi karfi a cikin Specc1l mutant neural folds (Fig. 5L da K), yana nuna cewa canje-canjen AJ sun fara.A cikin micrographs na lantarki na sassan kwanyar kwanyar embryos na E9.5, mun sake lura da ƙarar tabo mai yawa a cikin embryos na Specc1l idan aka kwatanta da WT (Fig. 5AA, BB da S1E-H).A hade, waɗannan sakamakon suna goyan bayan sakamakonmu na in vitro a cikin sel SPECC1L-kd U2OS kuma suna ba da shawarar cewa lalatawar AJ ta gabace siginar CNCC a cikin mutant embryos.
Ganin sanannen dangantakar adawa tsakanin ayyukan AKT da kwanciyar hankali na E-cadherin,17,28 mun yi hasashen shigar da siginar PI3K-AKT.Bugu da kari, mun lura da blistering subpidermal a cikin wasu mutant embryos da suka tsere daga mutuwa (<5%) a E9.5-10.5 kuma a maimakon haka zauna a kusa da E13.5 (Fig. S3).Subepidermal vesicles alama ce ta rage siginar PI3K-AKT dangane da PDGFRα12.Fantauzzo et al.(2014) ya ba da rahoton cewa rushewar PDGFRa na tushen PI3K kunnawa a cikin PdgfraPI3K/PI3K mutant embryos yana haifar da subepidermal vesicles, lahani na bututu na jijiyoyi, da ɓangarorin ɓangarorin ɓarke ​​​​.Lallai, matakan pan-AKT da phosphorylated Ser473-AKT mai aiki sun ragu a cikin vivo a cikin ƙwayoyin mutant na Specc1l zuwa kama E9.5 na amfrayo (Fig. 6A-D).Rage matakan phosphorylated Ser473-AKT na iya kasancewa gaba ɗaya saboda raguwar matakan pan-AKT a cikin vivo (FIG. 6E) da in vitro (FIG. 6F).An lura da raguwar in vitro ne kawai lokacin da ƙwayoyin U2OS suka haɗu da ƙarfi tare da canje-canje a cikin siffar tantanin halitta da yawan AJ (Hoto 6D).Don haka, bayananmu suna ba da shawarar cewa SPECC1L sabon labari ne mai daidaitawa na PI3K-AKT sigina a cikin ƙwayoyin cuta na craniofacial.
(A-E) E8.5 (A, B) da E9.5 (C, D) sassan kwanyar ko E9.5 lysates daga Specc1l mutant embryos (E) yana nuna matakan aiki na phosphorylated S473-AKT da pan-AKT Protein ragewa. , idan aka kwatanta da sarrafa WT.An yi lalata da yammacin Turai akan nau'in lysates na daji da lysates na mutant a ƙarƙashin yanayi iri ɗaya.Hotunan da aka nuna na SPECC1L an ɗauko su daga buge ɗaya.An cire wannan tabo kuma an sake gwada shi tare da anti-pan-ACT da β-actin antibodies.Matakan Pan-AKT a cikin folds na E8.5 (A, B) da matakan phosphorylated S473-AKT a cikin sassan kwanyar E9.5 sun ragu sosai.(F) Hakanan an rage matakan Pan-AKT a cikin lysates na ƙwayoyin SPECC1L-kd U2OS da aka girbe a babban haɗuwa.Kuskuren sanduna suna wakiltar SEMs daga ƙididdiga masu zaman kansu uku na Yamma.(GJ) Sassan embryos na WT a E9.5 masu tabo tare da KI67 da tsagewar caspase 3, bi da bi, suna nuna yaduwar tantanin halitta (G, G') da ƙaramin aikin apoptotic (H, H').Specc11 mutant embryos suna nuna kwatankwacin haɓakar tantanin halitta (I), amma adadin ƙwayoyin da ke jurewa apoptosis yana ƙaruwa sosai (J).
Sa'an nan kuma muka bincika alamun yaduwa da apoptosis.Ba mu lura da wani bambanci ba a cikin yaduwar embryos na E9.5 (Fig. 6E, G idan aka kwatanta da I) tare da ma'auni na yaduwa na 82.5% don maye gurbin WT da 86.5% don Specc1l mutants wanda aka auna ta KI67 tabo (p <0.56, Fisher's ainihin gwaji).Hakazalika, ba mu lura da wani bambanci a cikin apoptosis da aka auna ta hanyar tabo don tsagewar caspase 3 a cikin folds na jijiyoyi a E8.5 har sai an kama amfrayo (ba a nuna ba) (ba a nuna ba).Sabanin haka, apoptosis ya karu sosai a cikin duk E9.5 mutant embryos (Fig. 6F, H da J).Wannan haɓakar gabaɗaya a cikin apoptosis ya yi daidai da rage siginar PI3K-AKT da farkon mutuwar mahaifa29,30,31.
Na gaba, don tabbatar da rawar da ke haifar da siginar PI3K-AKT a cikin canje-canjen AJ a cikin ƙwayoyin kd ɗinmu, mun canza hanyar sinadarai cikin sarrafawa da ƙwayoyin kd (Hoto 7A-F).Mun yi amfani da matsayin alama canjin siffar tantanin halitta da aka gani a cikin sel SPECC1L-kd masu haɗaka, waɗanda muka ƙididdige su ta amfani da ma'auni mafi tsayi (tsawon tsayi) zuwa daidaitaccen girma na tsaye (nisa).Ana sa ran rabon 1 don ingantacciyar sel zagaye ko cuboidal (Hoto 7G).Bugu da ƙari ga siffar tantanin halitta, mun kuma tabbatar da tasiri akan AJ ta hanyar β-catenin tabo (Fig. 7A'-F').Hana hanyar PI3K-AKT ta amfani da wortmannin ya isa ya canza siffar tantanin halitta a cikin sel masu sarrafawa (Hoto 7A,C) da AJ (Hoto 7A').PI3K-AKT activator SC-79 bai shafi siffar tantanin halitta (FIG. 7A, E) ko fadada AJ (FIG. 7A') a cikin sel masu sarrafawa.A cikin ƙwayoyin SPECC1L-kd, ƙarin ƙaddamar da hanyar PI3K-AKT ya haifar da ƙara yawan apoptosis (Fig. 7B, D) da kuma karuwa mai girma a cikin β-catenin tabo (Fig. 7B'), daidai da mu a cikin vivo nauyi mutants.Mahimmanci, kunna hanyar PI3K-AKT ta inganta ingantaccen siffar tantanin halitta (Hoto 7B,F) da kuma AJ phenotypes (Hoto 7B”).Canje-canje a cikin siffar tantanin halitta an ƙididdige su azaman ƙimar zagaye na tantanin halitta (CCR) kuma idan aka kwatanta da mahimmanci kamar yadda aka bayyana a sama (FIG. 7G).Lalle ne, a cikin sel masu sarrafawa (Fig. 7G, CCR = 1.56), maganin wortmannin ya isa ya canza siffar tantanin halitta (Fig. 7G, CCR = 3.61, p <2.4 × 10-9) har zuwa daidai da wanda aka lura. Saukewa: SPECC1L.-kd Kwayoyin (Fig. 7G, CCR = 3.46).Maganin Wortmannin na kwayoyin SPECC1L-kd (Fig. 7G, CCR = 3.60, rashin kuskure) ba shi da mahimmanci fiye da kwayoyin kd da ba a kula da su ba (Fig. 7G, CCR = 3.46, rashin kuskure) ko kwayoyin kula da wortmannin (Fig. 7G)., CCR = 3.46, negligible) bugu da žari yana rinjayar haɓakar ƙwayar sel (7G, CCR = 3.61, maras kyau).Mafi mahimmanci, SC-79 AKT activator ya mayar da elongated phenotype na SPECC1L-kd Kwayoyin (Fig. 7G, CCR = 1.74, p <6.2 × 10-12).Waɗannan sakamakon sun tabbatar da cewa SPECC1L yana daidaita siginar PI3K-AKT kuma yana ba da shawarar cewa raguwar matsakaici a cikin SPECC1L yana rinjayar mannewar tantanin halitta, yayin da raguwa mai ƙarfi ke haifar da apoptosis (Fig. 8).
(A-F') Sarrafa (A, C, E) da SPECC1L-kd (B, D, F) kwayoyin da aka bi da su tare da PI3K-AKT hanya mai hana wortmannin (C, D) ko SC-79 activator (E, F) Jiyya Kwayoyin kulawa da ba a kula da su ba su ne cuboidal (A) tare da launi na β-cat na yau da kullum (A'), yayin da ƙwayoyin kd suna elongated (B) tare da ƙara yawan β-cat (B').Bayan danne hanyar PI3K-AKT, sel masu sarrafawa sun haɓaka (C) tare da haɓaka β-cat (C'), yayin da ƙwayoyin kd suka fara ɗaukar apoptosis (D), kama da embryos ɗin mu masu canzawa sosai kuma suna nuna ingantaccen β-cat.tabo (D').Bayan kunna hanyar PI3K-AKT, sel masu sarrafawa sun kasance cuboidal (E) kuma suna da β-cat (E') na yau da kullun, yayin da ƙwayoyin kd sun nuna ingantaccen siffar tantanin halitta (F) da β-cat (F'), yana nuna alamar tabo. (G) An ƙididdige matakin canjin siffar tantanin halitta a cikin (AF) ta amfani da ma'aunin zagaye na salula (CCR) mafi tsayi (tsawon) da madaidaicin girma na tsaye (nisa) ta amfani da software na MetaMorph.Kwayoyin da ba a kula da su ba (NT) SPECC1L-kd (CCR = 3.46) sun fi tsayi fiye da kwayoyin sarrafawa (CCR = 1.56, p <6.1 × 10-13).Hanawar Wort na hanyar PI3K-AKT a cikin sel masu sarrafawa ya isa ya haifar da irin wannan elongation a cikin siffar tantanin halitta (CCR=3.61, p<2.4 × 10-9).Hakanan, kunna AKT ta SC-79 a cikin ƙwayoyin SPECC1L-kd sun dawo da haɓakar tantanin halitta zuwa matakan sarrafawa (CCR = 1.74, p <6.2 × 10-12).Maganin Wortmannin na kwayoyin SPECC1L-kd ya haifar da karuwar apoptosis amma ba a kara karuwa a cikin canjin siffar tantanin halitta (CCR = 3.60) idan aka kwatanta da kd da ba a kula da su ba (CCR = 3.46, ns) ko kwayoyin kula da wortmannin (3.61) da aka lura a cikin.ns = ba komai.+/- Ana nuna ma'aunin SEM na sel 50.An ƙididdige bambance-bambancen ƙididdiga ta amfani da t-gwajin Student.
(A) Tsarin tsari na hanawa da kunna hanyar PI3K-AKT wanda ke haifar da canje-canjen AJ da ceto, bi da bi.(B) Samfurin da aka tsara don tabbatar da furotin AKT ta SPECC1L.
Premigratory CNCCs na buƙatar AJ lysis don rabuwa daga jijiyoyi na baya na neuroepithelial sel1,15,32.Ƙara tabo na abubuwan AJ da asarar apical-basal AJ asymmetric rarraba a cikin SPECC1L-raunan sel duka biyu a cikin vitro da in vivo, hade tare da kusancin jiki na SPECC1L zuwa β-catenin, suna ba da shawarar cewa ayyukan SPECC1L don kula da kwanciyar hankali na gida na AJ daidai. tsokoki na kungiya.maganin cytoskeleton.Ƙungiyar SPECC1L tare da cytoskeleton na actin da β-catenin da karuwa a cikin yawan adadin actin filaments a cikin rashin SPECC1L ya dace da karuwa a cikin yawan AJ.Wata yuwuwar ita ce ƙara yawan adadin actin fibers a cikin sel marasa ƙarancin SPECC1L yana haifar da canji a cikin tashin hankali na intercellular.Saboda damuwa na salula yana rinjayar tasirin AJ 33, canje-canjen ƙarfin lantarki na iya haifar da ƙarin yaduwa AJ 34 .Don haka duk wani canje-canje zai shafi matakan CNCC.
Wnt1 ana bayyana shi a farkon folds na jijiyoyi waɗanda ke haifar da ƙwayoyin jijiyoyi.Don haka, Wnt1-cre bin diddigin zuriyar yana nuna alamun farko da ƙaura NCC35.Koyaya, Wnt1 kuma yana alamar clones na nama na ƙwanƙwasa shima wanda aka samo daga farkon folds 35,36, yana mai yuwuwar tabon mu na E9.5 ga alamomin Wnt1 a cikin folds na jijiyoyi ba CNCC ba.Kyakkyawan tabon mu ga alamomin NCC AP2A da SOX10 sun tabbatar da cewa ɓoyayyen jijiyoyi na Specc11 mutant embryos sun ƙunshi CNCC.Bugu da ƙari, tun da AP2A da SOX10 sune alamomi na ƙaura na farko na NCC, tabbataccen tabo ya nuna cewa waɗannan sel sune CNCC bayan hijira waɗanda ba za a iya daidaita su ta hanyar E9.5 ba.
Bayananmu sun ba da shawarar cewa ka'idojin kwayoyin AJ ta SPECC1L ana yin sulhu ta hanyar siginar PI3K-AKT.An rage siginar AKT a cikin ƙananan ƙwayoyin SPECC1L da kyallen takarda.Sakamakon Fantauzzo et al.goyi bayan rawar kai tsaye don siginar PI3K-AKT a cikin ƙwayoyin cuta na craniofacial.(2014) ya nuna cewa rashin kunna siginar PDGFRa na tushen PI3K-AKT yana haifar da ɓarna phenotype.Mun kuma nuna cewa hanawa hanyar PI3K-AKT ya isa ya canza siffar AJ da tantanin halitta a cikin ƙwayoyin U2OS.Daidai da bincikenmu, Kayinu et al.37 ya nuna cewa raguwar tsarin PI3K α110 a cikin sel na endothelial yana haifar da irin wannan karuwa a cikin lalata β-catenin na pericellular, wanda ake magana a kai a matsayin karuwa a cikin "indexing index".Koyaya, a cikin ƙwayoyin endothelial waɗanda actin filaments an riga an tsara su sosai, danne hanyar PI3K-AKT yana haifar da sako-sako da sifar tantanin halitta.Sabanin haka, ƙwayoyin SPECC1L-kd U2OS sun nuna siffar tantanin halitta mai tsayi.Wannan bambanci na iya zama takamaiman nau'in tantanin halitta.Duk da yake kashe siginar PI3K-AKT na dindindin yana shafar cytoskeleton na actin, ana ƙaddara tasirin sifar tantanin halitta ta hanyar canje-canje a cikin tashin hankali wanda ya haifar da canje-canje a cikin yawa da ƙungiyar filayen actin na tsakiya.A cikin ƙwayoyin U2OS, mun yi amfani da canje-canjen siffar tantanin halitta kawai azaman alamar SPECC1L-rashin AJ canji da murmurewa.A ƙarshe, muna tsammanin cewa hana hanyar AKT a cikin rashi SPECC1L yana ƙaruwa da kwanciyar hankali na AJ kuma yana rage ƙaddamarwa a cikin CNCC.
Abin sha'awa, an rage matakan pan-AKT a cikin vitro da in vivo ban da matakan phosphorylated 473-AKT a cikin rashin SPECC1L, yana ba da shawarar ka'idojin PI3K-AKT siginar a matakin kwanciyar hankali na furotin AKT ko juyawa.Kwayoyin SPECC1L da MID1, dukansu suna hade da ciwo na Opitz / GBBB, suna ɓoye sunadaran da ke daidaita microtubules 18,22.Hanyar da SPECC1L da MID1 ke daidaita microtubule stabilization ba a fahimta sosai ba.A cikin yanayin SPECC1L, wannan ƙarfafawa ya haɗa da haɓakar acetylation na wani yanki na microtubules 18.Yana yiwuwa SPECC1L yana amfani da irin wannan tsari don daidaita sauran sunadaran kamar AKT.An nuna cewa acetylation na ragowar lysine a cikin furotin AKT yana haifar da raguwa a cikin mahallin membrane da phosphorylation38.Bugu da ƙari, ana buƙatar ƙaddamar da sarkar K63 a cikin ragowar lysine a kan AKT don ƙaddamar da membrane da kunnawa39,40.Daga cikin abubuwa da yawa da ke hulɗa tare da sunadaran SPECC1L da aka gano a cikin nau'ikan yisti mai girma iri-iri biyu-matasan fuska, huɗu - CCDC841, ECM2942, APC da UBE2I43 - an haɗa su a cikin jujjuyawar furotin ko kwanciyar hankali ta hanyar rarrabawa ko tarawa.SPECC1L na iya shiga cikin gyare-gyare na bayan fassarorin AKT lysine, yana shafar kwanciyar hankali AKT.Duk da haka, muhimmiyar rawar SPECC1L a cikin wuri da kwanciyar hankali na furotin AKT ya kasance da za a bayyana shi.
Mummunan lahani a cikin maganganun SPECC1L a cikin vivo sun haifar da ƙarar alamar AJ da lahani mai rufi na CNCC, da kuma ƙara yawan apoptosis da farkon mutuwar amfrayo.Rahotannin da suka gabata sun nuna cewa ƙwayoyin linzamin kwamfuta tare da ƙarin matakan apoptosis suna da alaƙa da lahani na bututun jijiyoyi 44,45,46,47 da lahani na craniofacial48.An ba da shawarar cewa mutuwar tantanin halitta mai yawa a cikin ɓangarorin jijiyoyi ko ɓangarorin pharyngeal na iya haifar da ƙarancin adadin ƙwayoyin da ake buƙata don ingantaccen motsi na morphogenetic 48,49,50.Sabanin haka, ƙananan layukan ƙwayoyin mu na SPECC1L tare da matsakaicin ragi na SPECC1L ya nuna canje-canjen AJ kawai ba tare da shaidar ƙarar mutuwar tantanin halitta ba.Koyaya, hana sinadarai na hanyar PI3K-AKT a cikin waɗannan ƙwayoyin Kd sun haifar da haɓakar apoptosis.Don haka, matsakaicin raguwa a cikin magana ko aiki na SPECC1L yana tabbatar da rayuwar tantanin halitta.Wannan ya yi daidai da lura da cewa embryos na Specc11 da ba kasafai suke tserewa kamawa a St.E9.5-watakila saboda raguwar haɓakar haɓakar ƙwayoyin cuta-suna iya rufe bututun jijiyoyi kuma suna tsayawa daga baya a cikin ci gaba, sau da yawa tare da lahani na craniofacial (Fig. S3).Hakanan ya yi daidai da wannan shine abin da ba a sani ba na heterozygous Specc1l embryos tare da rashin daidaituwa na craniofacial-watakila saboda haɓakar haɓakar ƙwayoyin halitta-da kuma ganowa a cikin zebrafish wanda ɗayan ɗayan SPECC1L guda biyu (specc1lb) ke haifar da ƙarancin amfrayo na phenotypes, gami da hasara na haifuwa. ƙananan muƙamuƙi da ɓangarorin biyu51.Don haka, heterozygous SPECC1L sauye-sauye-sauye-sauyen aiki da aka gano a cikin marasa lafiya na mutum na iya haifar da ƙananan lahani a cikin aikin SPECC1L a lokacin craniofacial morphogenesis, wanda ya isa ya bayyana ɓangarorin orofacial.Tsarin tushen SPECC1L na lambobin sadarwa na iya kuma iya taka rawa a cikin palatogenesis da haɗuwa da arches na pharyngeal.Ƙarin nazarin aikin SPECC1L zai taimaka wajen bayyana rawar da ake yi na lambobin sadarwa na wucin gadi a cikin CNCC yayin rufewar bututun jijiyoyi a cikin ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar ƙwayar cuta.
U2OS osteosarcoma iko da sel SPECC1L-kd an kwatanta su a baya (Saadi et al., 2011).Kwayoyin rigakafi akan SPECC1L suma an siffanta su a baya (Saadi et al., 2011).Anti-β-catenin rigakafi (zomo; 1:1000; Santa Cruz, Dallas, TX) ( linzamin kwamfuta; 1: 1000; Fasahar siginar salula, 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; Fasahar Siginar salula, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA) ) , KI67 (1: 1000; Fasahar Siginar salula, Danvers, MA), tsagewar caspase 3 (1:1000; Fasahar siginar salula, Danvers, MA) da β-actin (1:2500; Sigma-Aldrich, St. Louis, MO) an yi amfani da shi kamar yadda aka bayyana..Actin filaments an lalata su da Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado).
Kwayoyin kula da U2OS da sel SPECC1L-kd an haɓaka su cikin daidaitaccen babban glucose na DMEM wanda aka haɓaka tare da 10% ƙwayar ƙwayar tayi (Technology Life, Carlsbad, CA).Don canje-canjen AJ, 2 x 105 kwayoyin sun kasance a kan gilashin da aka bi da su tare da 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) kuma an lura da canje-canje a cikin siffar tantanin halitta.An tattara sel a wurare daban-daban da aka nuna: 4 hours bayan shuka (t = 1), sa'o'i 24 bayan shuka (t = 2), haɗuwa ba tare da canji a siffar tantanin halitta ba (t = 3), canza siffar tantanin halitta (t = 4). , 24 h bayan canjin siffar tantanin halitta (t = 5) da 48 h bayan canjin siffar tantanin halitta (t = 6) (Fig. 1, 2, 3).Don daidaita hanyar PI3K-AKT, an haɓaka sel a cikin abubuwan da aka nuna tare da mai hana PI3K-AKT wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) ko SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).An canza matsakaicin da ke ɗauke da sinadarai kowace rana.
An yi rikodin firam-by-frame akan sarrafa rayuwa da ƙwayoyin KD a ƙarƙashin yanayin al'ada na al'ada, kuma an tattara hotunan bambancin lokaci kowane minti 10 na kwanaki 7.An samo hotuna ta amfani da na'ura mai jujjuyawar Leica DM IRB mai sarrafa kwamfuta sanye take da matakin inji da makasudin 10 × N-PLAN da aka haɗa da kyamarar QImaging Retiga-SRV.A lokacin hoto, ana kiyaye al'adun tantanin halitta a 37 ° C a cikin yanayi mai laushi tare da 5% CO2.
Layukan kwayar halitta guda biyu na ES cell DTM096 da RRH048 daga Cibiyar Albarkatun Mutant Mouse Resource (UC Davis, CA) an yi amfani da su don samar da layukan linzamin kwamfuta na Specc11, wanda aka keɓance Specc1lgtDTM096 da Specc1lgtRRH046.A taƙaice, sel 129/REJ ES an allurar su cikin fashewar fashewar C57BL6.Sakamakon berayen chimeric na maza an haife su tare da mice C57BL6 mata don gano zuriya masu launin gashi na agouti.An yi amfani da kasancewar abubuwan da ake sakawa na tarkon kwayoyin halitta don gano heterozygotes.An ajiye beraye akan gaurayawan bango na 129/REJ;C57BL6.An tabbatar da wurin da aka shigar da vector tarkon kwayoyin halitta ta RT-PCR, jerin kwayoyin halitta, da haɓakar kwayoyin halitta (Ƙarin Hoto 1).Don gano layin CNCC na mice heterozygous Specc1lGT sau biyu, ROSAmTmG (#007576) da Wnt1-Cre (#003829) mice (Jackson Laboratory, Bar Harbor, ME) an ketare don samar da ROSAmTmG da Wnt1-Cre allele a Speccyol.Dukkan gwaje-gwajen da aka yi a cikin beraye an yi su ne bisa ga ka'idojin da Cibiyar Kula da Dabbobi ta Cibiyar Kula da Dabbobi ta Jami'ar Kansas Medical Center ta amince.
An gyara embryos a cikin (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) na 60 min a zafin jiki.Bayan gyare-gyare a cikin maganin tabo X-gal (5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP-40, 1 mg / ml X-gal) An gudanar da haɓaka tabo a 37 ° C. .° C a cikin sa'o'i 1-6.An gyara embryos a cikin 4% PFA kuma an gani.
Don lalatawar Yamma, an lulluɓe ƙwayoyin sel a cikin buffer lysis (Promega, Fitchburg, WI) wanda aka haɓaka tare da cakuda masu hana hana hana cutar HALT (Sigma-Aldrich, St. Louis, MO).An sarrafa Lysates akan 12% polyacrylamide Mini-PROTEAN TGX shirye-shiryen gels (Bio-Rad, Hercules, CA) kuma an tura su zuwa membranes na Immobilon PVDF (EMD Millipore, Billerica, MA).An toshe membranes a cikin 5% madara a cikin PBS mai dauke da 0.1% Tween.An sanya ƙwayoyin rigakafi a cikin dare a 4 ° C ko na sa'a daya a zazzabi na ɗaki.An yi amfani da Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) don tsara sigina.Don rigakafin rigakafi, embryos an gyara su cikin dare a cikin 4% PFA/PBS kuma an kiyaye su.An katange nama cryosections a cikin PBS dauke da 1% al'ada goat serum (Thermo Scientific, Waltham, MA) da 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) sa'an nan incubated a 4 ° C a cikin wani incubator a lokacin dare.tare da anti-antibody da fluorescent antibody na biyu (1:1000) na awa 1 a 4°C.An sanya sassan da ba su da kyau a cikin matsakaicin zinare na ProLong (Thermo Scientific, Waltham MA) kuma an sami hotuna masu lebur ta amfani da na'urar hangen nesa ta Leica TCS SPE.An yi kowane rigakafin rigakafi azaman gwaje-gwaje masu zaman kansu guda uku akan cirossection na aƙalla embryo na mutant guda biyu.An nuna gwajin wakilci.
An shigar da kwayoyin halitta a cikin buffer RIPA da aka gyara (20 mM Tris-HCl, pH 8.0, 1% NP-40, 130 mM NaCl, 10% glycerol, 2 mM EDTA, da HALT protease inhibitor (Sigma-Aldrich, St. Louis, MO) A taƙaice, an riga an tsarkake lysates tare da furotin G Magnetic beads (Life Technologies, Carlsbad, CA) sannan kuma a sanya su cikin dare a 4 ° C. tare da anti-SPECC1L ko IgG protein beads an yi amfani da su don cire SPECC1L kuma an yi amfani da gogewar Yamma ta amfani da anti -β-catenin antibody da aka bayyana a sama Gwajin haɗin gwiwar IP da aka nuna wakilcin gwaje-gwaje masu zaman kansu guda huɗu.
Kafaffen sel masu al'ada ko kyallen jikin linzamin kwamfuta an ba su zuwa cibiyar microscope na lantarki a Cibiyar Kiwon Lafiya ta Jami'ar Kansas.A taƙaice, an haɗa samfuran a cikin resin EMbed 812 (Electron Microscope Sciences, Fort Washington, PA), polymerized dare ɗaya a 60 ° C, kuma an raba shi a 80 nm ta amfani da Leica UC7 ultramicrotome sanye take da ruwan lu'u-lu'u.An hango sassan sassan ta amfani da JEOL JEM-1400 microscope na watsa wutar lantarki sanye da bindigar Lab6 mai nauyin 100kV.
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