ASTM A790 2507 / 2205 1.4462 / 1.4410 Duplex Welded Tube For Chemical Industry chemical components, erughị eru nke SPECC1L na-eduga n'ịkwụsi ike nke nkwonkwo gbawara agbawa na mbelata mwụda nke sel crest cranial.

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

Ụgwọ nke ụlọ ọrụ Liaocheng Sihe SS Material Co., Ltd.bụ onye na-emepụta ihe bụ ọkachamara na igwe anaghị agba nchara, ọkpọkọ annealed na-egbuke egbuke, tubing na-enweghị ntụpọ wdg.Iji mee ka ndị ahịa dị mfe, anyị nwekwara ọkpọkọ na ọkpọkọ welded.Ụgwọ nke ụlọ ọrụ Liaocheng Sihe SS Material Co., Ltd.nwere akụrụngwa mmepụta na nnwale kachasị elu.Anyị nwere ike imeju ihe ị chọrọ.Dabere na ọkọlọtọ siri ike, ọkpọkọ nke anyị mepụtara na-enwe nnabata OD na WT ziri ezi.Njikwa nnabata bụ nke ọma dabere n'ịmepụta ụkpụrụ.Ngwaahịa anyị na-enwe afọ ojuju mgbe niile na ndị ahịa.Ndị ahịa zụrụ ngwaahịa anyị mepụtara uru karịa.
a) OD ( dayameta elu): 3.18mm ruo 101.6mm
b) WT (Mgbidi ọkpụrụkpụ): 0.5mm ruo 20mm
c) Ogologo: Dị ka ndị ahịa chọrọ
d) Ụkpụrụ : ASTM A312;ASTM A269;ASTM A789;ASTM A790 wdg
e) Usoro usoro: ERW, EFW wdg

Ihe mmịfe na-egosi akụkọ atọ kwa slide.Jiri bọtịnụ azụ na nke na-esote ịkwaga na slides, ma ọ bụ bọtịnụ njikwa slide na njedebe ka ịgafe na slide ọ bụla.
Mkpụrụ ndụ akwara cranial (CNCC) na-ewepụ mpịaji akwara embrayo wee kwaga n'akụkụ oghere nke pharyngeal, bụ nke na-etolite ọtụtụ n'ime akụkụ etiti.Ngwọta CNCC na-arụ ọrụ dị mkpa na etiology nke orofacial cleft, ihe na-adịghị mma na-emekarị.Achọpụtala mmụgharị Heterozygous SPECC1L n'ime ndị ọrịa nwere mgbawa na-adịghị ahụkebe na syndromic.N'ebe a, anyị na-akọ na agba agba agba nke canonical adhesive junction (AJ), β-catenin na E-cadherin n'ime mkpụrụ ndụ knockdown SPECC1L, na micrographs eletrọn na-egosi mgbasa ozi apical-basal nke AJ.Iji ghọta ọrụ SPECC1L na craniofacial morphogenesis, anyị mepụtara ụdị òké ụkọ Specc1l.Mutants homozygous na-egbu nwa embrayo ma gosipụta mmechi akwara akwara na lamination CNCC.A na-abawanye ntụpọ protein AJ na mpịachi akwara mutant.Nrụrụ AJ a dabara na ntụpọ dị na delamination CNCC, chọrọ mgbasa AJ.Na mgbakwunye, Specc11 mutant ebelatala akara ngosi PI3K-AKT yana mmụba apoptosis.Na vitro, mgbochi dị nro nke PI3K-AKT na-egosi na sel ụdị anụ ọhịa zuru oke iji mee mgbanwe AJ.Nke dị mkpa, mgbanwe AJ nke SPECC1L na-akụtu nwere ike gbanwee site na ịgbalite ụzọ PI3K-AKT.Ejikọtara ọnụ, data ndị a na-atụ aro na SPECC1L, dị ka onye na-ahụ maka akara ngosi PI3K-AKT na bayoloji AJ, achọrọ maka mmechi akwara akwara yana stratification CNCC.
Cranial neural crest cell (CNCCs) na-abanye na neuroectoderm dorsal wee kewapụ na neuroepithelium nke akwara akwara na-eto eto site na usoro metụtara mgbanwe epithelial-mesenchymal (EMT) 1,2,3.CNCC na-ebuga epithelial na-akpaghasị njikọ intercellular wee ghọọ CNCC mesenchymal na-akwaga nke na-ejupụta arches pharyngeal nke mbụ na nke abụọ ma na-etolite ọtụtụ n'ime cartilage craniofacial.Ya mere, mkpụrụ ndụ ihe nketa na-achịkwa ọrụ CNCC na-akụghasịkarị na etiology nke craniofacial congenital anomalies dị ka orofacial clefts, na-emetụtakarị ọmụmụ 1/800 na US naanị.Otu n'ime nrụrụ a mụrụ8.
Delamination nke CNCC dabara na mmechi nke akwara akwara azụ n'etiti ụbọchị 8.5 na 9.5 nke mmepe embrayo na ụmụ oke.Mmụba nke ọtụtụ mkpụrụ ndụ ihe nketa orofacial na-akpakọrịta na-egosipụtakwa ụfọdụ ụdị ntụpọ akwara akwara, gụnyere Irf69,10, Ghrl310, Cfl111, na Pdgfrα12.Otú ọ dị, usoro nke mmechi tube neural na CNCC stratification nwere ike na-ewere onwe ya, dị ka Splotch mutant òké (Pax3) na-egosipụta ntụpọ na neural tube mmechi na-enweghị mmetụta ọ bụla na CNCC stratification ma ọ bụ Mbugharị 13,14.Ụdị òké ndị ọzọ nwere ntụpọ na dissection CNCC na mmechi tube neural ga-enyere aka ịkọwapụta ihe ndabere mkpụrụ ndụ nkịtị nke usoro abụọ a.
Mwepu nke CNCC site na sel neuroepithelial na-achọ ka mgbaze nke njikọ nrapado (AJs), nke mejupụtara ogige protein nwere, n'etiti ndị ọzọ, E-cadherin, β-catenin, α-E-catenin, na α-actinin jikọtara ya na actin filaments 2. Ọmụmụ ihe gbasara oke okwu E-cadherin na mpịachi akwara gosipụtara mbelata ma ọ bụ igbu oge na delamination CNCC.N'aka nke ọzọ, nkwụsị nke E-cadherin na-ebute stratification mbụ15,16.Ọtụtụ n'ime ihe ndị na-emezi EMT n'oge stratification CNCC bụ ihe ntụgharị (AP2a, Id2, FOXD3, SNAIL, TWIST, SOX10) na extracellular matrix (ECM) na-edozi protein dị ka matrix metalloproteinases (MMPs), agbanyeghị CNCC bụ ndị na-achịkwa cytoskeletal AJ kpọmkwem. amabeghị.A mara ụzọ PI3K-AKT ka ọ na-emegide ọkwa E-cadherin, ọkachasị site na nyocha kansa17.Nnyocha ndị e mere n'oge na-adịbeghị anya egosila na mfu nke PI3K-AKT na-egosi PDGFa na ụmụ oke na-eduga n'ọdịdị craniofacial, gụnyere mgbawa palate na neural tube ntụpọ12.Agbanyeghị, mmekọrịta dị n'etiti ụzọ PI3K-AKT yana nkwụsi ike AJ na stratification CNCC amabeghị.
Anyị na-achọpụtabu SPECC1L dị ka mkpụrụ ndụ mutant mbụ n'ime mmadụ abụọ nwere mgbawa siri ike na-esi n'ọnụ ruo n'anya, nke a maara dị ka oblique cleft (ObFC) ma ọ bụ Tessier IV18 cleft.Achọpụtala mmụgharị SPECC1L n'ime ezinụlọ abụọ dị iche iche nwere ọrịa autosomal Opitz G/BBB (OMIM #145410), nke ndị mmadụ metụtara gosipụtara hyperdistance na egbugbere ọnụ / palate19, yana n'otu ezinụlọ nwere ọrịa Tibi overdistance (OMIM #145420)20. .Ihe karịrị ọkara nke ọrịa Opitz G/BBB bụ X-njikọ (OMIM #300000) ma kpatara mmụgharị dị na mkpụrụ ndụ MID1, nke na-etinye protein 22 nke skeleton cell metụtara microtubule.Anyị na-eche na SPECC1L, bụkwa protein jikọtara ya na microtubules na actin cytoskeleton, nwere ike na-edozi akara ngosi achọrọ maka nhazigharị cytoskeleton actin n'oge adhesion cell na mbugharị 18.Site na in vitro na vivo ọmụmụ, anyị na-akọwa ugbu a SPECC1L dị ka akwụkwọ akụkọ regulator nke AJ kwụsie ike site PI3K-AKT akara.N'ọkwa cellular, ụkọ SPECC1L mere ka mbelata ọkwa nke protein pan-AKT na mmụba na mgbasa nke apical-basal nke AJ, bụ nke a kpochapụrụ site na ntinye kemịkalụ nke ụzọ AKT.Na vivo, embrayo na-ezughị ezu Specc11 na-egosi mmechi tube akwara na-adịghị mma yana mbelata nkesa CNCC.Ya mere, SPECC1L na-arụ ọrụ na nrịbama sel adhesion na-achịkwa nke ukwuu chọrọ maka ọrụ CNCC nkịtị n'oge morphogenesis ihu.
Iji kọwapụta ọrụ SPECC1L na ọkwa cellular, anyị jiri eriri osteosarcoma cell kwụsiri ike nke U2OS akọwara na mbụ na SPECC1L18.Ndị a kwụsiri ike U2OS mkpụrụ ndụ na SPECC1L (kd) knockdown nwere a agafeghị oke (60-70%) mbelata na ọkwa nke SPECC1L transcripts na protein, tinyere ntụpọ na Mbugharị na nhazigharị nke actin cytoskeleton 18. N'ụzọ dị iche, a siri ike na-agafe agafe mbelata na. E gosiputara SPECC1L na-eduga na ntụpọ mitotic 23.N'ịkọwapụta n'ihu, anyị chọpụtara na mkpụrụ ndụ SPECC1L-kd anyị kwụsiri ike gbanwere morphology n'ọkwa dị elu nke nnabata (Foto 1).Selụ njikwa n'otu n'otu na sel kd na nhụta dị ala yiri (Foto 1A, D).Mgbe awa 24 gachara, mkpụrụ ndụ nchịkwa na-ejigide ọdịdị cuboid ha (Fig 1B, E), ebe SPECC1L-kd sel elongated (Fig 1C, F).Oke mgbanwe a n'ọdịdị cell weghaara na vivo ndụ onyonyo nke mkpụrụ ndụ njikwa na mkpụrụ ndụ kd (ihe nkiri 1).Iji chọpụta ọrụ SPECC1L na sel confluent, anyị buru ụzọ nyochaa okwu ya.Anyị chọpụtara na ọkwa protein SPECC1L mụbara n'elu ngwakọta (Njirimara 1G), ebe ọkwa ederede SPECC1L abawanyeghị (Njirimara 1H).Tụkwasị na nke ahụ, ka njupụta cell na-abawanye, protein SPECC1L na-akwakọba na njedebe intercellular (Fig 2A-E), nke nwere ụkpụrụ na-ejikọta ya na nke β-catenin na-ejikọta akpụkpọ ahụ (Fig. 2A'-E').Nyere njikọ nke SPECC1L na actin cytoskeleton 18,23 anyị chere na SPECC1L na-emekọrịta ihe na njikọ nrapado dabere na actin (AJ).
(AF) SPECC1L knockdown (DF) mkpụrụ ndụ na-agbatị na nnukwu confluence (F) ma e jiri ya tụnyere sel U2OS (AC).Egosiri ebe a bụ atọ n'ime isi oge isii (T1, T3, T6) nke anyị họọrọ maka njupụta cell dị iche iche.(G) Nyocha nke Western blot na-egosi na protein SPECC1L kwụsiri ike n'ọkwa dị elu ma e jiri ya tụnyere obere ọkwa nnabata na sel njikwa.Western blot nke SPECC1L na-egosi band 120 kDa a na-atụ anya ya na igwe dị arọ dị elu, enwere ike gbanwee ntụgharị asụsụ (*).A na-eme nyocha nke Western blot n'okpuru otu ọnọdụ maka mkparịta ụka dị ala na nke dị elu.Foto ndị na-egosi SPECC1L na nhụta dị ala na nke dị elu ka ewepụtara n'otu ntụpọ ahụ.E wepụrụ otu ntụpọ ahụ wee jiri ọgwụ mgbochi β-actin nyochaa ya ọzọ.(H) Nyocha RT-PCR nke ọnụọgụ egosighi mgbanwe dị ịrịba ama na ọkwa ederede SPECC1L.Ogwe mperi na-anọchite anya SEM site na nnwale anọ nwere onwe.
(AE) Anyị họọrọ isi oge isii (T1-T6) na-anọchite anya ọnụ ọgụgụ dị iche iche nke cell iji mee ka nyocha ọdịdị nke cell na mgbanwe AJ na mkpụrụ ndụ U2OS na SPECC1L knockdown (kd).Ise nke mbụ n'ime oge ndị a gụnyere otu mkpụrụ ndụ (T1), 50-70% ngwakọta nke obere ụyọkọ cell (T2), ngwakọta na-enweghị nhazigharị kd cell (T3), imegharị mkpụrụ ndụ kd (T4), na mgbanwe elekere 24.n'ụdị azụ nke sel kd (T5).A na-agbasasị protein nke SPECC1L na cytoplasm na T1 (A), mana a hụrụ nchikota ya na oke intercellular n'oge na-esote (B-E, akụ).(FJ) β-catenin na-egosi nchịkọta yiri nke ahụ na oke intercellular jikọtara ya na mgbagwoju AJ.(A'-E') SPECC1L na β-catenin na-egosi ncha nchacha na oke cell na njupụta cell dị elu (akụ).(F'-J') N'ime mkpụrụ ndụ SPECC1L-kd, ntụpọ β-catenin na-egosi na ọ dị mma na njupụta cell dị ala (F'-H'), mana gbasaa ka ọdịdị sel na-agbanwe (I', J'; akụ), na-egosi na AJ agbanweela.Ogwe = 10µm.
Anyị nwara ịchọpụta mmetụta erughị eru SPECC1L na AJ.Anyị na-eji ọtụtụ akara akara AJ, gụnyere canonical components F-actin, myosin IIb, β-catenin, na E-cadherin24,25,26,27.Actin stress fibers mụbara na mkpụrụ ndụ SPECC1L-kd dị ka akọwara na mbụ (Fig 3A, B) 18.Myosin IIb jikọtara ya na actin filaments gosipụtara mmụba yiri nke ahụ na sel SPECC1L-kd na vitro (Fig 3C, D).β-catenin jikọtara AJ na-ejikọta cadherin na akpụkpọ ahụ cell, na-egosi usoro okwu okwu "mmanụ aṅụ" nkịtị na cubocytes nchịkwa (Fig 3E, G).N'ụzọ na-akpali mmasị, na ihe oyiyi dị larịị na-eji microscopy confocal, β-catenin (Fig. 3E, F) na E-cadherin (Fig 3G, H) na-agbapụta na akpụkpọ ahụ cell nke confluent SPECC1L-deficient sel gosipụtara ụkpụrụ ndị a ma ama nke ogologo ntụpọ.Mgbasawanye nke β-catenin na-ejikọta AJ na mkpụrụ ndụ kd bụ nke a na-akpọkarị na mgbagwoju anya, ma ọ pụtara na ọ ga-ebute mgbanwe na ọdịdị cell (Fig 2F-J, F'-J').Iji chọpụta ọdịdị anụ ahụ nke ntụpọ AJ gbatịrị agbatị, anyị nyochara oke mkpụrụ ndụ n'elu ala apical-basal nke mkpụrụ ndụ SPECC1L-kd U2OS site na nnyefe elektrọn microscopy (TEM) (Nyocha 3I, J).N'adịghị ka sel njikwa (Fig 3I), nke nwere mpaghara dị iche iche nke electron dị iche iche na-egosi AJ (akụ), mkpụrụ ndụ kd (Fig 3J) gosipụtara mpaghara buru ibu, nke na-ejikọta ọnụ nke nnukwu njupụta electron na-egosi AJ tinyere ụgbọ elu apicobasal..Na mgbakwunye, na akụkụ transverse, anyị hụrụ nnukwu mpịachi cell folds na kd cell (Fig. S1A, B), nke na-akọwa ogologo usoro nke β-catenin na E-cadherin staining bands (Fig. 3F, H).N'ịkwado ọrụ SPECC1L na AJ, β-catenin jikọtara ya na SPECC1L na lysates nke mkpụrụ ndụ U2OS confluent (Fig 3K).Tinyere ịgbatị immunostaining maka akara AJ, nyocha TEM kwekọrọ n'echiche anyị na ụkọ SPECC1L na-abawanye njupụta AJ apical-basal na ọdịiche.
(AH) Mmụba nke F-actin n'ime sel kd na awa 48 post-fusion (T6; A, B).Ntụcha myosin IIb gbanwere na F-actin (C, D).Ụdị dị mma nke β-catenin na E-cadherin akpụkpọ anụ na-agbapụta na sel nchịkwa (E, G) ka emelitere na sel SPECC1L-kd (F, H).Ogwe = 10 µm.(I-J) micrographs eletrọn na-elele njikọ intercellular apical-basal.Selụ njikwa na-egosi mpaghara elektrọn dị iche iche na-egosi njikọ nnyapade (I, akụ).N'ụzọ dị iche, njikọ ahụ dum apical-basal dị na sel SPECC1L-kd pụtara njupụta eletrọn (J, akụ), na-egosi ụbara njupụta na mgbasa nke njikọ nrapado.(K) β-catenin jikọtara ya na SPECC1L na lysates cell U2OS confluent.Onyonyo ewepụtara site na otu ntụpọ na-anọchi anya otu n'ime nnwale onwe anọ.
Iji ghọta ọrụ SPECC1L na craniofacial morphogenesis, anyị kere Specc1l deficient mouse model site na iji ahịrị ES trap cell abụọ nwere onwe ha, DTM096 na RRH048 (BayGenomics, CA), nke na-anọchite anya intron 1 na Specc1l transcripts weghaara na 15 (Fig 1) .4A, foto S2).Ekpebiri ọnọdụ genomic nke ntinye vector decoy site na usoro genome dum wee kwado ya site na PCR (Fig S2).Nhazi ọnyà mkpụrụ ndụ abụọ ahụ kwekwara ka njikọta nke ndị nta akụkọ Specc11-lacZ mgbe ejidere ya.Ya mere, a na-eji okwu lacZ kpebisiri ike site na ntụpọ X-gal dị ka ihe ngosi nke okwu Specc11.Abụọ alleles gosipụtara ụkpụrụ okwu lacZ yiri nke ahụ, yana ọnyà mkpụrụ ndụ DTM096 na intron 1 na-egosi okwu siri ike karịa RRH048 na intron 15 (egosighi ya).Agbanyeghị, a na-egosipụta Specc1l n'ọtụtụ ebe, yana ngosipụta siri ike na mpịaji akwara na E8.5 (Nyocha 4B), n'ime akwara akwara na usoro ihu na E9.5 na E10.5 (Nyocha 4C, D), yana n'ịzụlite aka. na E10.5 na anya (Foto 4D).Anyị na-akọ na mbụ na okwu SPECC1L na akpa pharyngeal mbụ na E10.5 dị na epithelium na mesenchyme18 dị n'okpuru, kwekọrọ na usoro CNCC.Iji nwalee okwu SPECC1L na CNCC, anyị mere E8.5 neural folds (Figure 4E-J) na E9.5 skull sections (Figure 4K-).Na E8.5, SPECC1L nwere ntụpọ akwara nke ọma (Fig 4E, H), gụnyere mkpụrụ ndụ nwere akara NCC (Fig. 4G, J).Na E9.5, SPECC1L (Fig. 4K, N) siri ike na-akwagharị CNCC na-ejikọta ya na AP2A (Fig. 4L, M) ma ọ bụ SOX10 (Fig. 4O, P).
(A) Ngosipụta atụmatụ nke mouse Specc11 gene na-egosi ntinye vector decoy na ES DTM096 (intron 1) na RRH048 (intron 15) clones cell.(BD) ntụpọ lacZ nke heterozygous Specc1lDTM096 embrayo na-anọchi anya okwu Specc1l sitere na E8.5 ruo E10.5.NE = neuroectoderm, NF = akwara akwara, PA1 = akpa pharyngeal mbụ.(EP) SPECC1L immunostaining na NCC akara AP2A na SOX10 na E8.5 (NF; EJ) neural folds na E9.5 (KP) akụkụ okpokoro isi.A na-ahụta ntụpọ SPECC1L n'ọtụtụ ebe na mpịaji akwara E8.5 (E, H; arrowheads), gụnyere mkpụrụ ndụ akpọrọ AP2A (F, G; isi akụ) na SOX10 (I, J; isi akụ).Na E9.5, SPECC1L nwere ntụpọ siri ike na-akwaga CNCC (K, N; akụ) akpọrọ AP2A (L, M; akụ) na SOX10 (O, P; akụ).
Ịgafe n'etiti heterozygous Specc1lDTM096/+ na Specc1lRRH048/+ ụmụ oke na-egosi na ọnyà abụọ nke mkpụrụ ndụ ihe nketa anaghị ejikọta ya na heterozygotes na-emepụta ihe na homozygotes embrayo maka ma ọnyà ọnyà a na-egbu nwa (Table S1).Ọnụọgụ Mendelian gosipụtara mbelata nke ịdị ndụ nke heterozygotes n'oge ọmụmụ (atụ anya 1.34 vs. 2.0).Anyị chọpụtara na ọnwu dị ala n'etiti heterozygotes, ụfọdụ nwere anomalies craniofacial (Fig. S3).Agbanyeghị, nnabata dị ala nke ụdị ụdị craniofacial ndị a na-eme ka o sie ike ịmụ usoro pathophysiological ha dị n'okpuru.Ya mere, anyị lekwasịrị anya na phenotype embrayo na-egbu egbu nke mutant homozygous Specc11.
Ọtụtụ ogige heterozygous ma ọ bụ homozygous Specc1lDTM096/RRH048 mutant embrayo etoliteghị mgbe E9.5-10.5 (Fig 5A-D), na akwara akwara adịghị emechi n'ihu (Fig 5B, D) na mgbe ụfọdụ mechiri azụ (egosighi ya). ..Mmechi mmechi akwara akwara cranial a jikọtara ya na imirikiti CNCC akara DLX2 fọdụrụ na mpịaji akwara na E10.5, na-egosi enweghị dissection (Ọnụ ọgụgụ 5A'-D').Iji chọpụta ma ọ bụrụ na a belatara nha CNCC n'ozuzu ya, anyị kpadoro ahịrị CNCC na GFP na ahịrị ọnyà mkpụrụ ndụ anyị na Wnt1-Cre na ROSamTmG.Anyị na-ebunye usoro GFP+ NCC na GFP- (RFP+) na-abụghị NCC sitere na embrayo dum.Na E9.5, oke nke CNCC nke akpọrọ GFP na-asọpụta agbanweghị nke ọma n'etiti WT na embrayo mutant (egosighi), na-egosi nkọwa CNCC nkịtị.Ya mere, anyị chere na ihe fọdụrụ Wnt1-Cre na DLX2 ntụpọ na oghere akwara ekpughere (Njirimara 5B') bụ n'ihi nhazi CNCC nwere ntụpọ, ikekwe n'ihi ụbara njupụta ma ọ bụ mgbasa nke sel AJ, dịka a hụrụ na sel SPECC1L-kd.Anyị na-eji akara NCC SOX10, AP2A, na DLX2 iji kwado ọnụnọ nke CNCC n'ime oghere akwara (Nyocha 5E-R).Na E8.5, a na-ahụ ntụpọ neural fold maka ihe nrịbama NCC atọ na ngalaba nke WT (Fig. 5E, G, I) na Specc1l mutant (Fig. 5F, H, J).Na E9.5, ebe NCC nrịbama nwere ntụpọ NCC na-akwaga na ngalaba WT (Fig. 5M, O, Q), a na-ahụ ntụpọ NCC fọdụrụnụ na mpịachi akwara nke Specc1l mutant embrayo (Fig. 5N, P, R).N'ihi na SOX10 na DLX2 akara CNCC na-akwaga, nsonaazụ a na-egosi na CNCC enweghị SPECC1L na-enweta nkọwapụta n'oge njem njem mana ha anaghị akwaga na mpịaji akwara.
Enweghị Specc11 na-eduga na mmechi akwara akwara nwere nkwarụ, delamination nke mkpụrụ ndụ crest neural neural na AJ.
(A, B') E9.5 WT (A) Embrayo bu sel crest neural neural (CNCC) nke akpọrọ Wnt1-Cre (A').N'ụzọ dị iche, embrayo Specc11 mutant na-egosi oghere akwara mepere emepe (B), isi akụ) na CNCC ndị na-akwagabeghị (B', isi akụ).(C, D') Onyonyo ubi na-egbuke egbuke (C, D') na immunostaining (C', D') nke akara CNCC DLX2 nke E10.5 WT embrayo (C, C') na Specc1l (D, D').N'ime embrayo WT E10.5, DLX2-nke CNCC na-achịkwa gill arches (C', akụ), ebe n'ime mutant, ntụpọ pụtara ìhè na-adịgide na oghere oghere oghere (D', akụ) na na arches pharyngeal mbụ (D', akụ).) na ụfọdụ ntụpọ (akụ) na-egosi adịghị mma delamination na Mbugharị nke CNCC.ER) Akụkụ nke WT na Specc1l mutant embrayo na ọkwa E8.5 (E-L) na E9.5 (M–R) ka ejiri akara NCC SOX10 (E, F, M, N), AP2A (G, H, O, P) na DLX2 (I, J, Q, R).Na E8.5, a na-ahụ ntụpọ NCC n'ụdị anụ ọhịa anụ ọhịa (NF) na ngalaba mutant.Njikọta nke SOX10 na β-catenin na E8.5 WT (K) na mutant (L) gosipụtara mmụba β-catenin na-abawanye na sel sel na mpịachi akwara.Na E9.5, a na-ahụ ntụpọ ụdị anụ ọhịa nke CNCC na-akwaga (M, O, Q), ebe na mutants, CNCC na-enweghị ntụpọ nwere ntụpọ oghere oghere oghere (N, P, R).(S–Z) Nyocha akara akara vivo AJ na ngalaba coronal nke WT na Specc11DTM096/RRH048 embrayo nwere ngbanwe E9.5.Egosiri ụgbọ elu ngalaba ihe dị ka n'akụkụ aka nri elu.N'akụkụ nke anụ ahụ mutant, a hụrụ mmụba nke F-actin (S, T) na myosin IIb (U, V).Yiri nsonaazụ in vitro dị na Fig. 3, n'ime embrayo mutant, emelitere akpụkpọ anụ maka β-catenin (W, X) na E-cadherin (Y, Z).(AA-BB) Igwe okwu eletrọn nke akụkụ nke ẹmbrayo ụdị anụ ọhịa na-ele anya n'ofe oke nke cell apical-basal na-egosi mpaghara elektrọn dị iche iche nke na-egosi njikọ nrapado (AA, akụ).N'ụzọ dị iche, na ngalaba nke Specc11 mutant embrayo (BB, akụ), njikọ apicobasal dum bụ nnukwu elektrọn, na-egosi njupụta na mgbasawanye nke nrapado.
Iji nwalee echiche anyị na ibelata oyi akwa bụ n'ihi mgbanwe AJ, anyị nyochara akara AJ na mpịachi akwara ekpughere nke embrayo mutant Specc1l (Fig. 5S-Z).Anyị hụrụ na-abawanye na actin stress fibers (Fig. 5S, T) na concomitant ụba localization nke myosin IIB ntụpọ na actin fibers (Fig. 5U, V).N'ụzọ dị mkpa, anyị hụrụ mmụba nke β-catenin (Fig. 5W, X) na E-cadherin (Fig 5Y, Z) na njedebe intercellular.Anyị nyochakwara ntụpọ β-catenin nke NCC na mpịachi akwara nke embrayo E8.5 (Fig. 5K, L).β-catenin staining pụtara na ọ siri ike na Specc1l mutant neural folds (Fig 5L na K), na-atụ aro na mgbanwe AJ amalitela.N'ime micrographs eletrọn nke akụkụ okpokoro isi nke embrayo E9.5, anyị hụkwara ọzọ mmụba eletrọn na-agbasa n'ime embrayo mutant Specc1l ma e jiri ya tụnyere WT (Fig. 5AA, BB na S1E-H).Ejikọtara ọnụ, nsonaazụ ndị a na-akwado nsonaazụ in vitro anyị na sel SPECC1L-kd U2OS wee tụọ aro na ntụpọ AJ na-adịghị mma na-ebute stratification CNCC n'ime embrayo anyị.
Nyere mmekọrịta mmegide ama ama n'etiti ọrụ AKT na nkwụsi ike E-cadherin, 17,28 anyị chere na ntinye aka nke akara ngosi PI3K-AKT.Na mgbakwunye, anyị hụrụ ọnya subepidermal n'ụfọdụ n'ime embrayo anyị nke gbanarịrị ọnwụ (<5%) na E9.5-10.5 kama biri na gburugburu E13.5 (Fig. S3).Subepidermal vesicles bụ akara ngosi PI3K-AKT belatara dabere na PDGFRα12.Fantauzzo et al.(2014) kọrọ na nkwụsị nke PDGFRa dabeere PI3K ịgbalite na PdgfraPI3K/PI3K mutant embrayo na-ebute vesicles subepidermal, ntụpọ tube neural, na phenotypes palate phenotypes.N'ezie, ọkwa nke pan-AKT na phosphorylated Ser473-AKT na-arụ ọrụ na-ebelata na vivo na Specc1l mutant tissues na E9.5 embrayo arrest (Fig. 6A-D).Mbelata ọkwa nke phosphorylated Ser473-AKT nwere ike ịbụ kpamkpam n'ihi mbelata ọkwa pan-AKT na vivo (FIG. 6E) na in vitro (FIG. 6F).A hụrụ mbelata in vitro naanị mgbe mkpụrụ ndụ U2OS nwere njikọ siri ike yana mgbanwe ọdịdị sel yana njupụta AJ (Njirimara 6D).Ya mere, data anyị na-atụ aro na SPECC1L bụ ezigbo onye na-achịkwa PI3K-AKT na-egosi na craniofacial morphogenesis.
(A-E) E8.5 (A,B) na E9.5 (C,D) ngalaba okpokoro isi ma ọ bụ E9.5 lysates sitere na Specc1l mutant embrayo (E) na-egosi ọkwa nke phosphorylated S473-AKT na pan-AKT Protein Mbelata. , tụnyere njikwa WT.A na-eme nhichapụ Western na ụdị lysates anụ ọhịa na mutant lysates n'okpuru otu ọnọdụ ahụ.Ihe onyonyo egosiri maka SPECC1L ka ewepụtara n'otu ntụpọ.E wepụrụ otu ntụpọ ahụ wee jiri ọgwụ mgbochi pan-ACT na β-actin nyochaa ya ọzọ.A na-ebelata ọkwa Pan-AKT na E8.5 neural folds (A, B) na ọkwa nke phosphorylated S473-AKT na ngalaba okpokoro isi E9.5.(F) A na-ebelata ọkwa Pan-AKT n'otu aka ahụ na lysates nke mkpụrụ ndụ SPECC1L-kd U2OS ewetere na nnukwu nnabata.Ogwe mperi na-anọchite anya SEMs sitere na nha atọ nke Western blot nwere onwe.(GJ) Akụkụ nke WT embrayo na E9.5 nwere ntụpọ KI67 na caspase 3 gbawara agbawa, n'otu n'otu, na-egosi mmụba cell (G, G') na obere ọrụ apoptotic (H, H').Specc11 mutant embrayo na-egosi mmụba cell yiri nke ahụ (I), mana ọnụọgụ nke mkpụrụ ndụ na-anata apoptosis na-abawanye nke ukwuu (J).
Anyị na-enyocha ihe nrịbama nke mmụba na apoptosis.Anyị ahụghị ihe ọ bụla dị iche na mmụba nke embrayo E9.5 (Fig. 6E, G ma e jiri ya tụnyere I) na ntinye mmụba nke 82.5% maka mutant WT na 86.5% maka Specc1l mutants tụrụ site na KI67 staining (p <0.56, Fisher's). ule ziri ezi).N'otu aka ahụ, anyị ahụghị ihe ọ bụla dị iche na apoptosis tụrụ site na nchacha maka caspase 3 gbawara agbawa na mpịachi akwara na E8.5 ruo mgbe embrayo jidere (egosighi ya) (egosighi ya).N'ụzọ dị iche, apoptosis abawanyela nke ukwuu na embrayo E9.5 mutant niile (Fig 6F, H na J).Mmụba a n'ozuzu ya na apoptosis kwekọrọ na mbelata akara ngosi PI3K-AKT na egbu egbu nwa embrayo n'oge29,30,31.
Na-esote, iji kwado ọrụ kpatara maka akara ngosi PI3K-AKT na mgbanwe AJ na sel kd anyị, anyị jiri kemịkalụ gbanwee ụzọ njikwa yana mkpụrụ ndụ kd (Ọnyonyo 7A-F).Anyị na-eji dị ka akara mkpụrụ ndụ mgbanwe ọdịdị phenotype hụrụ na confluent SPECC1L-kd mkpụrụ ndụ, nke anyị jiri ọnụ ọgụgụ nke ndị kasị ogologo akụkụ (ogologo) ka kwekọrọ vetikal akụkụ (obosara).A na-atụ anya oke nke 1 maka sel gburugburu ma ọ bụ cuboid (Figure 7G).Na mgbakwunye na ọdịdị cell, anyị kwadoro mmetụta na AJ site na β-catenin staining (Fig. 7A'-F').Mgbochi nke ụzọ PI3K-AKT site na iji wortmannin zuru ezu iji gbanwee ọdịdị sel na sel njikwa (Njirimara 7A,C) na AJ (Nyocha 7A').PI3K-AKT activator SC-79 emetụtaghị ọdịdị sel (FIG. 7A, E) ma ọ bụ mgbasawanye AJ (FIG. 7A') na sel njikwa.Na mkpụrụ ndụ SPECC1L-kd, nkwụsịkwu nke ụzọ PI3K-AKT mere ka apoptosis dịkwuo elu (Fig 7B,D) na mmụba dị ịrịba ama na β-catenin staining (Fig. 7B'), na-adabere na anyị in vivo arọ mutants.N'ụzọ dị mkpa, ịgbalite ụzọ PI3K-AKT mere ka ọdịdị sel dịkwuo mma nke ukwuu (Ọnọdụ 7B,F) na AJ phenotypes (Figure 7B").A na-atụle mgbanwe n'ụdị cell dị ka cell roundness ratio (CCR) ma jiri ya tụnyere mkpa dị ka akọwara n'elu (FIG. 7G).N'ezie, na sel nchịkwa (Fig 7G, CCR = 1.56), ọgwụgwọ wortmannin zuru oke iji gbanwee ọdịdị nke cell (Fig 7G, CCR = 3.61, p <2.4 × 10-9) ruo n'ókè yiri nke a hụrụ. na SPECC1L.-kd mkpụrụ ndụ (Fig. 7G, CCR = 3.46).Ọgwụgwọ Wortmannin nke mkpụrụ ndụ SPECC1L-kd (Fig. 7G, CCR = 3.60, negligible) abụghị ihe dị ịrịba ama karịa mkpụrụ ndụ kd na-adịghị edozi (Fig. 7G, CCR = 3.46, negligible) ma ọ bụ wortmannin-mere sel (Fig. 7G)., CCR = 3.46, negligible) na-emetụtakwa elongation cell (7G, CCR = 3.61, negligible).Nke kachasị mkpa, SC-79 AKT activator weghachiri phenotype elongated nke sel SPECC1L-kd (Fig. 7G, CCR = 1.74, p <6.2 × 10-12).Nsonaazụ ndị a na-akwado na SPECC1L na-achịkwa akara ngosi PI3K-AKT ma na-atụ aro na mbelata nke ukwuu na SPECC1L na-emetụta adhesion cell, ebe mbelata siri ike na-eduga na apoptosis (Fig 8).
(A-F') Njikwa (A, C, E) na SPECC1L-kd (B, D, F) mkpụrụ ndụ ejiri PI3K-AKT ụzọ inhibitor wortmannin (C, D) ma ọ bụ SC-79 activator (E, F) ọgwụgwọ. .Mkpụrụ ndụ nchịkwa na-adịghị agwọ ọrịa bụ cuboidal (A) na nkịtị β-cat cellular staining (A'), ebe kd cell na- elongated (B) na ụba β-cat staining (B').Mgbe emechibidoro ụzọ PI3K-AKT, mkpụrụ ndụ njikwa elongated (C) nwere mgbasa β-cat (C'), ebe mkpụrụ ndụ kd malitere ịmalite apoptosis (D), dị ka embrayo anyị gbanwere nke ukwuu ma na-egosi β-cat nke ukwuu.ntụpọ (D').Mgbe emechara ụzọ PI3K-AKT, mkpụrụ ndụ njikwa nọgidere na-enwe cuboidal (E) ma nwee ntụpọ β-cat (E') nkịtị, ebe mkpụrụ ndụ kd gosipụtara ọdịdị sel dị mma (F) na β-cat (F'), na-egosi ntụpọ. (G) A na-agbakọ ogo nke mgbanwe ọdịdị cell na (AF) site na iji usoro okirikiri cell (CCR) nke ogologo kachasị ogologo (ogologo) yana akụkụ vetikal kwekọrọ (obosara) site na iji ngwa MetaMorph.Mkpụrụ ndụ SPECC1L-kd anaghị agwọ (NT) (CCR = 3.46) dị ogologo karịa sel njikwa (CCR = 1.56, p <6.1 × 10-13).Mgbochi Wort nke ụzọ PI3K-AKT na sel njikwa zuru oke iji mee ka elongation yiri ya na ọdịdị sel (CCR = 3.61, p <2.4 × 10-9).N'otu aka ahụ, AKT ịgbalite site na SC-79 na sel SPECC1L-kd weghachiri elongation cell na ọkwa nchịkwa (CCR = 1.74, p <6.2 × 10-12).Ọgwụgwọ Wortmannin nke mkpụrụ ndụ SPECC1L-kd mere ka apoptosis dịkwuo elu ma ọ dịghị mmụba ọzọ na mgbanwe ọdịdị cell (CCR = 3.60) ma e jiri ya tụnyere kd na-adịghị agwọ ọrịa (CCR = 3.46, ns) ma ọ bụ wortmannin-agwọ ọrịa (3.61) hụrụ na .ns = ọ dịghị mkpa.+/- E gosipụtara nha SEM maka mkpụrụ ndụ 50.A gbakọọ ndịiche ọnụ ọgụgụ site na iji t-ule nwata akwụkwọ.
(A) Ngosipụta atụmatụ nke mgbochi na ịgbalite ụzọ PI3K-AKT na-ebute mgbanwe AJ na nnapụta, n'otu n'otu.(B) Nlereanya akwadoro maka nkwado protein AKT site na SPECC1L.
CNCC na-aga n'ihu chọrọ AJ lysis ka ọ kewapụ na sel neuroepithelial neural fold1,15,32.Mmụba ntụpọ nke AJ components na mfu nke apical-basal AJ asymmetric nkesa na SPECC1L-deficient cell ma na vitro na in vivo, jikọtara ya na anụ ahụ dị nso nke SPECC1L na β-catenin, na-atụ aro na SPECC1L na-arụ ọrụ iji nọgide na-enwe nkwụsi ike mpaghara AJ nke ọma. mọzụlụ nhazi.ọrụ nke cytoskeleton.Njikọ nke SPECC1L na actin cytoskeleton na β-catenin na mmụba nke ọnụ ọgụgụ actin filaments condensed na enweghị SPECC1L kwekọrọ na mmụba a hụrụ na njupụta AJ.Ihe ọzọ nwere ike ime bụ na ọnụọgụ actin na-abawanye na sel ndị na-enweghị SPECC1L na-eduga na mgbanwe na esemokwu intercellular.N'ihi na nrụgide cellular na-emetụta AJ 33 dynamics, mgbanwe voltaji nwere ike ime ka AJ 34 gbasaa karịa.Ya mere mgbanwe ọ bụla ga-emetụta ọkwa CNCC.
A na-egosipụta Wnt1 na mpịaji akwara mbụ nke na-ebute mkpụrụ ndụ crest neural.Ya mere, nchọta usoro usoro Wnt1-cre na-egosi ma tupu na ịkwaga NCC35.Agbanyeghị, Wnt1 na-akara akara clones ụbụrụ azụ azụ nke sitere na mpịaji akwara mbụ 35,36, na-eme ka ọ bụrụ ntụpọ anyị nke mutants E9.5 maka akara Wnt1 na mpịaji akwara mepere emepe abụghị CNCC.Nchacha anyị dị mma maka akara NCC AP2A na SOX10 gosipụtara na mpịachi akwara ekpughere nke embrayo mutant Specc11 nwere n'ezie CNCC.Na mgbakwunye, ebe AP2A na SOX10 bụ akara nke NCC na-akwaga n'oge, ntụpọ dị mma gosipụtara na mkpụrụ ndụ ndị a bụ CNCC na-akwaga mba ọzọ nke na-enweghị ike ịgbatị E9.5.
Ihe omuma anyi na-egosi na akara molikula nke AJ nke SPECC1L bu akara ngosi PI3K-AKT.A na-ebelata akara AKT na sel na anụ ahụ erughị eru SPECC1L.Nchọpụta nke Fantauzzo et al.na-akwado ọrụ kpọmkwem maka akara ngosi PI3K-AKT na craniofacial morphogenesis.(2014) gosiri na enweghị ike nke PDGFRa dabeere PI3K-AKT akara na-eduga na a chụrụ n'ọnụ phenotype.Anyị na-egosikwa na mgbochi nke ụzọ PI3K-AKT zuru ezu iji gbanwee ọdịdị AJ na cell na sel U2OS.N'ikwekọ na nchọpụta anyị, Ken et al.37 gosiri na nkwụsị nke PI3K α110 subnit na sel endothelial na-ebute mmụba yiri nke ahụ na pericellular β-catenin staining, nke a na-ezo aka dị ka mmụba na "ntụgharị njikọ njikọ".Agbanyeghị, na mkpụrụ ndụ endothelial nke actin filaments ahaziri nke ọma, nkwụsị nke ụzọ PI3K-AKT na-ebute ọdịdị sel rụrụ arụ.N'ụzọ dị iche, mkpụrụ ndụ SPECC1L-kd U2OS gosipụtara ọdịdị sel elongated.Ọdịiche a nwere ike bụrụ kpọmkwem ụdị cell.Ọ bụ ezie na nkwụsị nke akara ngosi PI3K-AKT na-emetụta cytoskeleton actin na-adịgide adịgide, a na-ekpebi mmetụta na ọdịdị sel site na mgbanwe na esemokwu kpatara mgbanwe na njupụta na nhazi nke etiti actin fibers.N'ime mkpụrụ ndụ U2OS, anyị na-eji naanị mgbanwe ọdịdị sel dị ka akara nke SPECC1L-deficient AJ mgbanwe na mgbake.N'ikpeazụ, anyị na-eche na mgbochi nke ụzọ AKT na ụkọ SPECC1L na-abawanye nkwụsi ike AJ ma belata delamination na CNCC.
N'ụzọ na-akpali mmasị, a na-ebelata ọkwa pan-AKT na vitro na in vivo na mgbakwunye na ọkwa 473-AKT phosphorylated na enweghị SPECC1L, na-atụ aro ụkpụrụ nke PI3K-AKT na-egosi na ọkwa nke AKT protein kwụsie ike ma ọ bụ ntụgharị.Mkpụrụ ndụ SPECC1L na MID1, ha abụọ jikọtara ya na ọrịa Opitz / GBBB, na-etinye protein ndị na-eme ka microtubules guzosie ike 18,22.Usoro nke SPECC1L na MID1 mediate microtubule stabilization aghọtachaghị nke ọma.N'ihe banyere SPECC1L, nkwụsi ike a gụnyere nkwalite acetylation nke akụkụ nke microtubules 18.Ọ ga-ekwe omume na SPECC1L na-eji usoro yiri ya mee ka protein ndị ọzọ dị ka AKT kwụsie ike.Egosiputara na acetylation nke lysine residues na protein AKT na-eduga n'ibelata n'ime mpaghara akpụkpọ ahụ na phosphorylation38.Tụkwasị na nke ahụ, a na-achọ ka a na-emepụta ihe nke K63 yinye n'otu ihe fọdụrụ na lysine na AKT maka nhazi nke akpụkpọ ahụ na ịgbalite39,40.N'ime ọtụtụ ihe na-emekọrịta ihe na protein SPECC1L achọpụtara na ihe dị iche iche dị elu na-emepụta yist abụọ-ngwakọ, anọ - CCDC841, ECM2942, APC na UBE2I43 - etinyela aka na ntụgharị protein ma ọ bụ nkwụsi ike site na njedebe ma ọ bụ sumoylation.SPECC1L nwere ike itinye aka na ngbanwe ntụgharị nke AKT lysine residues, na-emetụta nkwụsi ike AKT.Agbanyeghị, ọrụ dị oke egwu nke SPECC1L na nhazi na nkwụsi ike nke protein AKT ka ka ga-akọwapụta.
Nrụrụ siri ike na okwu SPECC1L na vivo butere mmụba nke akara AJ na ntụpọ CNCC nwere ntụpọ, yana mmụba apoptosis na ọnwụ embrayo n'oge.Akụkọ ndị gara aga egosila na ụmụ oke oke nwere oke apoptosis na-ejikọta ya na ntụpọ neural tube 44,45,46,47 na craniofacial ntụpọ48.Atụwo aro na ọnwụ mkpụrụ ndụ dị ukwuu na mpịachi akwara ma ọ bụ pharyngeal arches nwere ike ime ka ọnụ ọgụgụ sel ezughi oke achọrọ maka mmegharị morphogenetic kwesịrị ekwesị 48,49,50.N'ụzọ dị iche, ahịrị sel ụkọ SPECC1L anyị nwere okwu SPECC1L na-ebelata nke ọma gosipụtara naanị mgbanwe AJ na-enweghị ihe akaebe na-abawanye ọnwụ cell.Agbanyeghị, mgbochi kemịkalụ nke ụzọ PI3K-AKT na mkpụrụ ndụ Kd ndị a butere apoptosis mụbaa.Ya mere, mbelata agafeghị oke na okwu ma ọ bụ ọrụ SPECC1L na-eme ka ị dị ndụ cell.Nke a dabara na nlebanya na embrayo Specc11 na-adịghị ahụkebe na-agbanarị njide na St.E9.5-ikekwe n'ihi mbelata gene njide arụmọrụ-na-enwe ike mechie ha neural tubes na-akwụsị mgbe e mesịrị na mmepe, mgbe mgbe na craniofacial ntụpọ (Fig. S3).Ọzọkwa na nke a bụ ihe na-adịghị ahụkebe nke heterozygous Specc1l embrayo na craniofacial abnormalities-ikekwe n'ihi mmụba nke mkpụrụ ndụ ihe nketa-yana nchoputa na zebrafish nke otu n'ime abụọ SPECC1L orthologues (specc1lb) na-akpata mbubreyo phenotypes embrayo, gụnyere ọnwụ. agba dị ala na mgbawa abụọ51.Ya mere, heterozygous SPECC1L mmụgharị na-arụ ọrụ nke a na-achọpụta na ndị ọrịa mmadụ nwere ike ime ka obere mmebi dị na SPECC1L ọrụ n'oge craniofacial morphogenesis, zuru ezu iji kọwaa ihe mgbawa orofacial ha.Usoro dabere na SPECC1L nke kọntaktị intercellular nwekwara ike rụọ ọrụ na palatogenesis na ngwakọta nke arches pharyngeal.Nnyocha ndị ọzọ nke ọrụ SPECC1L ga-enyere aka ịkọwa ọrụ nke kọntaktị intercellular nwa oge na CNCC n'oge mmechi tube neural na neuroepithelial cell motility na craniofacial morphogenesis.
U2OS osteosarcoma njikwa na sel SPECC1L-kd ka akọwara na mbụ (Saadi et al., 2011).A marakwa ọgwụ mgbochi megide SPECC1L na mbụ (Saadi et al., 2011).Mgbochi mgbochi β-catenin (oke bekee; 1:1000; Santa Cruz, Dallas, TX) (òké; 1: 1000; Technology 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; Teknụzụ Mgbasa Ozi Cell, Danvers, MA), pan-AKT (1:1000; ThermoFisher Scientific, Waltham, MA) ) , KI67 (1:1000; Teknụzụ Mgbasa Ozi Cell, Danvers, MA), 3 caspase gbawara agbawa (1:1000; Teknụzụ Mgbasa Ozi Cell, Danvers, MA) na β-actin (1:2500; Sigma-Aldrich, St. Louis, MO ) ejiri mee ihe dịka akọwara..Eji Acti-stain rhodamine phalloidin (Cytoskeleton, Denver, Colorado) merụọ ihe ndị Actin filaments.
Azụlitere mkpụrụ ndụ njikwa U2OS na sel SPECC1L-kd na ọkwa glucose dị elu DMEM agbakwunyere na serum bovine nwa ebu n'afọ 10% (Life Technologies, Carlsbad, CA).Maka mgbanwe AJ, mkpụrụ ndụ 2 x 105 dị na iko na-emeso ya na 0.1% porcine gelatin (Sigma-Aldrich, St. Louis, MO) ma hụ maka mgbanwe na ọdịdị sel.A na-anakọta mkpụrụ ndụ n'oge dị iche iche egosipụtara: 4 awa mgbe ịgha mkpụrụ (t = 1), 24 awa mgbe mkpụrụ (t = 2), confluence na-enweghị mgbanwe na cell udi (t = 3), mgbanwe na cell udi (t = 4) , 24 h mgbe mgbanwe ọdịdị cell (t = 5) na 48 h mgbe mgbanwe ọdịdị cell (t = 6) (Fig. 1, 2, 3).Iji megharịa ụzọ PI3K-AKT, a na-emepụta mkpụrụ ndụ na ntinye ego egosipụtara na PI3K-AKT inhibitor wortmannin (TOCRIS Biosciences, Minneapolis, Minnesota) ma ọ bụ SC-79 activator (TOCRIS Biosciences, Minneapolis Adams, Minnesota).A gbanwere usoro nke nwere kemịkalụ kwa ụbọchị.
Emere ndekọ nke Frame-site-frame na njikwa ndụ yana mkpụrụ ndụ KD n'okpuru ọnọdụ omenala nkịtị, a na-anakọta ihe onyonyo nke oge ọ bụla nkeji iri ọ bụla maka ụbọchị 7.Enwetara onyonyo site na kọmpụta na-achịkwa Leica DM IRB nke nwere igwe nrụzi na ebumnobi 10 × N-PLAN jikọtara na igwefoto QImaging Retiga-SRV.N'oge eserese, a na-edobe omenala cell na 37ºC na ikuku iru mmiri nwere 5% CO2.
A na-eji ahịrị mkpụrụ ndụ abụọ ES cell DTM096 na RRH048 sitere na Regional Mutant Mouse Resource Center (UC Davis, CA) iji wepụta ahịrị òké pere mpe Specc11, ahapụtara Specc1lgtDTM096 na Specc1lgtRRH046.Na nkenke, a gbanyere mkpụrụ ndụ 129/REJ ES n'ime blastocysts C57BL6.Eji ụmụ oke ụmụ nwoke chimeric pụta na ụmụ oke C57BL6 iji mata ụmụ nwere agba akwa agouti.A na-eji ọnụnọ nke ọnyà mkpụrụ ndụ ihe nketa vector iji chọpụta heterozygotes.A na-edobe ụmụ oke na nzụlite agwakọta nke 129/REJ;C57BL6.A kwadoro ebe ntinye nke mkpụrụ ndụ ihe nketa ọnyà site na RT-PCR, usoro mkpụrụ ndụ ihe nketa, na mmeju mkpụrụ ndụ ihe nketa (Ntinye mgbakwunye 1).Iji chọpụta usoro ọmụmụ CNCC nke ụmụ oke heterozygous Specc1lGT okpukpu abụọ, ROSAmTmG (#007576) na Wnt1-Cre (#003829) ụmụ oke (Jackson Laboratory, Bar Harbor, ME) gafere iji mepụta ROSAmTmG na Wnt1-Cre allele in Speccyol.Emere nnwale niile n'ime ụmụ oke dịka usoro iwu nke Institutional Animal Care and Use Committee nke Mahadum Kansas Medical Center kwadoro.
A na-edozi embrayo na (1% formaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 0.02% NP-40, 5 mM EGTA) maka 60 min na ụlọ okpomọkụ.Mgbe edozichara na ngwọta ntụpọ 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) A na-eme mmepe ntụpọ na 37 ° C. .Celsius C n'ime awa 1-6.Edebere embrayo na 4% PFA wee hụ ya anya.
Maka ihichapụ nke ọdịda anyanwụ, a na-etinye mkpụrụ ndụ n'ime ihe nchekwa lysis na-agafe agafe (Promega, Fitchburg, WI) na ngwakọta nke ndị na-egbochi protease HALT (Sigma-Aldrich, St. Louis, MO).A na-ahazi Lysates na 12% polyacrylamide Mini-PROTEAN TGX gels njikere (Bio-Rad, Hercules, CA) ma bufee ya na akpụkpọ anụ Immobilon PVDF (EMD Millipore, Billerica, MA).E gbochiri akpụkpọ ahụ na mmiri ara ehi 5% na PBS nwere 0.1% Tween.A na-etinye ọgwụ mgbochi ọrịa n'abali na 4 Celsius ma ọ bụ otu awa na okpomọkụ ụlọ.Ejiri Femto SuperSignal West ECL reagent (Thermo Scientific, Waltham, MA) maka ọgbọ mgbama.Maka ịgba ọgwụ mgbochi ọrịa, embrayo na-edozi n'otu abalị na 4% PFA/PBS ma chekwaa ya.E gbochiri anụ ahụ cryosections na PBS nwere 1% nkịtị ewu serum (Thermo Scientific, Waltham, MA) na 0.1% Triton X-100 (Sigma-Aldrich, St. Louis, MO) wee tinye ya na 4 Celsius C na incubator n'oge okpomọkụ. abalị.nwere mgbochi mgbochi na fluorescent secondary antibody (1:1000) maka 1 hour na 4°C.Edebere akụkụ ndị nwere ntụpọ na ProLong gold media (Thermo Scientific, Waltham MA) wee nweta onyonyo dị larịị site na iji leica TCS SPE confocal microscope.Emere ọgwụ mgbochi ọ bụla dị ka nnwale atọ nọọrọ onwe ya na nhụsianya nke opekata mpe embrayo abụọ mutant.Egosiri nnwale onye nnọchi anya.
A na-etinye mkpụrụ ndụ n'ime ihe nchekwa RIPA gbanwere (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) Na nkenke, a na-eji protein G magnetik beads mee ka lysates dị ọcha (Life Technologies, Carlsbad, CA) wee tinye ya n'abali na 4 Celsius C na mgbochi SPECC1L ma ọ bụ IgG protein beads iji wepụ SPECC1L na Western blotting mere site na iji mgbochi mgbochi. -β-catenin mgbochi akọwara n'elu Nnwale ngalaba-IP egosiri bụ ihe nnọchianya nke nnwale anọ nwere onwe.
Enyere mkpụrụ ndụ emebere nke ọma ma ọ bụ anụ ahụ embrayo òké na etiti microscopy eletrọn na Mahadum Kansas Medical Center.Na nkenke, agbakwunyere ihe nlele na EMbed 812 resin (Electron Microscope Sciences, Fort Washington, PA), polymerized n'abali na 60 Celsius, wee kewaa ya na 80 nm site na iji Leica UC7 ultramicrotome nke nwere agụba diamond.A na-ahụ anya akụkụ site na iji microscope elektrọn nnyefe JEOL JEM-1400 nwere egbe Lab6 100 kV.
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