The authors would like to acknowledge Wensheng Liu for invaluable help with cell imaging. This work was supported by grants from the&#160;US National Institutes of Health (R01 CA182467), American Cancer Society (RSG-09-252-01-RMC), Lynn Sage Foundation, and A Sister&#8217;s Hope Foundation.

1 Cella Cultura della EMT induzione Nota: EMT può essere indotta da trattamenti di TGFβ o espressione ectopica di fattori di trascrizione Twist, Chiocciola, o Zeb1 / 2 in cellule epiteliali. Descritto in questo protocollo è un sistema EMT inducibile mediante espressione della proteina di fusione Twist-ER in cellule immortalizzate umane epiteliali mammarie (HMLE / Twist-ER, un dono di Dr. J Yang, UCSD) 9,11,29. Al 4-hydroxytamoxifen (TAM) il trattamento, la proteina di fusione Twist-ER trasloca nel nucleo di guidare la trascrizione e risultati in una transizione completa EMT in 12-14 giorni 9,11,29. Altri sistemi inducibili EMT, come HMLE / Snail-ER, HMLE / TGFβ, e MCF10A / TGFβ, si possono trovare nelle nostre precedenti pubblicazioni 11,30. <ol><li> Mantenere le cellule HMLE / Twist-ER in mammaria mezzo di crescita delle cellule epiteliali privo di siero (MEGM) in un incubatore a 37 ° C con il 5% di CO 2. Passage le cellule ogni 2-3 giorni quando raggiungono l&#39;80% di confluenza. Incubare le cellule con tripsina 0,15% a 37 ° C per 5-10 minuti, quindi inattivare la tripsina con siero di vitello al 5% / DMEM. Spin le cellule e risospendere in MEGM. Cellule portapiatti in un nuovo piatto di coltura tissutale con un rapporto di 1 a 4. </li><li> Sciogliere TAM in etanolo per ottenere una soluzione 200 mM. Proteggere TAM dalla luce e conservare a -20 ° C. Prima dell&#39;uso, appena aggiungere TAM in MEGM materie ad una diluizione 1: 10.000 per effettuare una concentrazione finale di 20 nM TAM. </li><li> Per l&#39;induzione di EMT, piastra cellule 1,6 x 10 6 HMLE / Twist-ER in un 10 centimetri piatto di coltura di tessuti in duplice copia. Celle di coltura con 20 supporti nM TAM-contenenti MEGM. </li><li> Piatto cellule 1,6 x 10 6 HMLE / Twist-ER in un piatto di 10 centimetri in duplice copia e il trattamento di cellule con 0,01% di etanolo come controllo non-TAM-trattati. </li><li> Due giorni dopo l&#39;incubazione, scattare foto sotto un microscopio ottico a 10X di ingrandimento per registrare cambiamenti morfologici delle cellule. </li><li> Aspirare il mezzo da un piatto di controllo o di TAM-trattaticellule. Lavare le cellule con PBS freddo e celle raccolto da demolizione metà della piastra in tampone di lisi RNA per l&#39;isolamento dell&#39;RNA e l&#39;altra metà in tampone RIPA per l&#39;analisi di proteine. </li><li> Cellule passaggio dal controllo o piatti TAM-trattati con 1,6 x 10 6 cellule ri-placcatura in un piatto di 10 centimetri in duplice copia. Continuamente trattare le cellule con 20 nM TAM o di un veicolo. </li><li> Ripetere i passaggi da 5 a 7 una volta ogni due giorni fino al giorno 14, in cui il tempo, le cellule Twist-ER TAM-trattati mostrano una mesenchimale morfologia fusiforme, mentre le cellule di controllo del veicolo trattate mantengono la morfologia epiteliale ciottoli-like. </li></ol> 2 Caratterizzazione di EMT Nota: A completamento della EMT è indicato da: (1) cambiamenti morfologici delle cellule da una morfologia epiteliale ciottoli come a un aspetto fibroblastica-come a forma di fuso; (2) Perdita di localizzazione E-caderina a giunzioni cellula-cellula; e (3) L&#39;espressione commutazione di marcatori EMT, definito dalla perditadi marcatori epiteliali e aumento dei marcatori mesenchimali. Cellulari cambiamenti morfologici vengono acquisite da microscopio ottico a 10X di ingrandimento nel corso del tempo di induzione EMT, descritto nella Sezione 1 Immuno-fluorescenza rilevamento di E-caderina in cellule-giunzioni è descritto in questa sezione. L&#39;espressione di markers di EMT viene rilevato mediante immunoblotting. Marcatori epiteliali comuni sono E-caderina, γ-catenina, e occludina, e marcatori mesenchimali includono fibronectina, N-caderina e vimentina. Procedure generali di immunoblotting sono descritti nella sezione 4. <ol><li> Al giorno 12 del trattamento TAM, semi di 1 x 10 5 cellule su un vetro coprioggetti circolare 12 millimetri disposte in un pozzetto di una piastra da 24 pozzetti. </li><li> 48 ore dopo che le cellule di semina, medie Aspirare e lavare con pre-riscaldato PBS. </li><li> Fissare le cellule con pre-riscaldato paraformaldeide 4% per 15 min. </li><li> Lavare le cellule tre volte con PBS. </li><li> Incubare le cellule con 0,2% Triton X-100 in PBS per 15minuti a temperatura ambiente. </li><li> Lavare le cellule tre volte con PBS. </li><li> Incubare le cellule a 5% BSA / PBS per 1 ora a temperatura ambiente per bloccare legame non specifico. </li><li> Incubare le cellule con l&#39;anticorpo E-caderina primario in una diluizione 1:50 in 1% BSA / PBS in una camera di umidificata O / N a 4 ° C. NOTA: L&#39;intervallo per la diluizione anticorpo primario è normalmente compreso tra 1:50 e 1: 200, e la diluizione ottimale per ogni anticorpo deve essere determinato sperimentalmente. </li><li> Decantare l&#39;anticorpo primario e lavare le cellule tre volte con PBS. </li><li> Incubare le cellule con l&#39;anticorpo secondario fluorescente marcato in una diluizione 1: 500 per 1 ora a temperatura ambiente. Da questo punto in poi, proteggere le cellule dalla luce. </li><li> Decantare l&#39;anticorpo secondario e lavare tre volte con PBS. </li><li> Cellule macchia con 0,1-1 g / ml DAPI o Hoechst per 10 min. </li><li> Lavare le cellule con PBS per tre volte. </li><li> Montare coprioggetto con una goccia di mezzo di montaggio, e coprioggetto tenuta con smalto. </li><li> Osservare le cellule e prendereimmagini sotto un microscopio a fluorescenza 63X. </li></ol> Colorazione con anticorpi supplementari può essere eseguita per confermare il fenotipo EMT. Ad esempio, N-caderina e α-actina del muscolo liscio possono essere colorati per un fenotipo mesenchimale 29,31,32. Riorganizzazione della struttura del citoscheletro può essere monitorato mediante colorazione delle cellule con falloidina per la F-actina 11. Inoltre, saggi di motilità cellulare e la resistenza delle cellule morte possono essere eseguite per esaminare le proprietà del EMT 11. 3 Rilevamento di Splice Isoforme Utilizzo qRT-PCR saggi <ol><li> Disegno Primer NOTA: Le coppie di primer deve essere accuratamente studiato per amplificare isoforme di splicing distinti. Exon skipping, l&#39;evento splicing alternativo più comunemente osservato, è illustrata qui come esempio per illustrare la strategia di progettazione di primer. Figura 1A mostra una quattro esone pre-mRNA, con il terzo esone come esone variabile. L&#39;inclusione dell&#39;esone 3 risultati inla produzione di un giunto isoforma più lunga, mentre skipping dell&#39;esone 3 genera una giuntura isoforma più corta. I primer devono essere progettati per distinguere l&#39;isoforma esone-3-incluso e esone-3-saltato isoforma, e per rilevare la trascrizione completa della mRNA. <ol><li> Per il rilevamento di inclusione dell&#39;esone, progettare con cura un primer in avanti all&#39;interno della variabile esone 3, e un primer reverse all&#39;interno della vicina esone costitutiva 4, permettendo l&#39;amplificazione specifica della variabile isoforma esone-incluso. Assicurarsi di non progettare sia in avanti e reverse primer all&#39;interno dello stesso esone variabile al fine di evitare i prodotti della PCR amplificati a partire dal DNA genomico o pre-mRNA. </li><li> Per amplificare specificamente esone eventi saltare, di design uno dei primer che abbracciano la giunzione dell&#39;esone costitutiva 2 e esone 4 che altrimenti sarebbe distrutto quando esone variabile 3 è incluso. Progettare l&#39;altro fondo all&#39;interno del esone costitutiva 4 Come tale, i prodotti di PCR vengono generati solo quando la variabile esone 3 è excluded e la costitutiva esone 2 e l&#39;esone 4 sono successivamente uniti. </li><li> Per rilevare le trascrizioni totali del gene, disegnare i primer entro due esoni costitutivi 1 e 2 Pertanto, i prodotti di PCR sono amplificati da tutte le isoforme. </li><li> Assicurarsi che la temperatura di fusione (Tm) per tutti i primer è di circa 55 ° C, la lunghezza di ogni primer è di circa 18-22 nucleotidi, e la dimensione dei prodotti di PCR è compresa tra 80 e 150 paia di basi. </li></ol></li><li> Estrazione di RNA: RNA estratto utilizzando un kit di totale isolamento di RNA (vedi tabella dei Materiali). <ol><li> Raccogliere le cellule in 350 microlitri di buffer di lisi RNA. </li><li> Aggiungere 350 ml di etanolo al 70% al lisato e mescolare accuratamente. </li><li> Trasferire il campione alla colonna di purificazione dell&#39;RNA. </li><li> Centrifugare a 10.000 xg per 1 min e scarto flow-through. </li><li> Lavare la colonna una volta con 500 ml di RNA di lavaggio buffer di I e due volte con RNA tampone di lavaggio II. </li><li> Rimuovere il tampone di lavaggio RNA residuoII dalla colonna mediante centrifugazione a velocità massima per 2 min. </li><li> Trasferire la colonna in una nuova provetta da 1,5 ml, aggiungere acqua priva di nucleasi 50 microlitri al centro della matrice colonna ed eluire l&#39;RNA per centrifugazione a velocità massima. </li></ol></li><li> Sintesi di cDNA First-strand <ol><li> Determinare la concentrazione e la qualità di RNA con la misura di assorbimento UV. NOTA: Buona RNA qualità dovrebbe avere un / 280 nm rapporto di assorbanza 260 nm di circa 2.0. </li><li> Impostare trascrittasi inversa (RT) reazioni che contengono 250 ng di RNA totale, 0,05 mg primer esameriche casuale, 50 pmol MgCl 2, 10 pmol dNTP, 2 microlitri di buffer 5 × GoScript, e 1 ml RT enzima in un volume finale di 20 ml. </li><li> Eseguire reazioni RT a 42 ° C per 1 ora, seguito da incubazione a 70 ° C per 5 minuti per inattivare l&#39;enzima RT. </li></ol></li><li> Real-time PCR <ol><li> Impostare 20 microlitri reazioni che consistono di 10 microlitri SYBR master mix verde,0.1-1.0 modello cDNA microlitri, e 5 pmol avanti e indietro primer. </li><li> Eseguire PCR in tempo reale con 40 cicli dei seguenti passi: 95 ° C denaturazione per 10 sec, 58 ° C ricottura per 10 sec e 60 ° C per 30 sec estensione. Eseguire reazioni PCR in triplice copia. </li><li> Controllare le curve di dissociazione e fare in modo che un singolo picco si osserva per ogni set di primer. </li><li> Ottenere i valori di ciclo di quantificazione (Cq) calcolando i secondi valori derivati ​​curve di amplificazione. </li><li> Calcolare la quantità relativa (RQ) del mRNA bersaglio in campioni indotto, rispetto al campione uninduced utilizzando il &#39;delta delta Cq (ΔΔCq)&#39; metodo come mostrato dalla seguente formula. Utilizzare geni housekeeping, come GAPDH o TBP, come riferimento. </li></ol></li></ol><img alt="Equazione 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Per calcolare con maggiore precisione i valori relativi delle isoforme di splicing, tegli usa di più geni di riferimento dovrebbe essere considerato. I risultati possono essere analizzati utilizzando un metodo di quantificazione del valore assoluto modificato descritto da Pfaffl et al 33,34. 4 Esame di Splice Isoforme a livello proteico <ol><li> Raccogliere le cellule in 100 microlitri di buffer RIPA ghiacciato. </li><li> Impostare lisati su ghiaccio per circa 10 min. </li><li> Centrifugare a 14000 g per 10 min a 4 ° C, e raccogliere il surnatante. </li><li> Misurare la concentrazione di proteine ​​mediante saggi Bradford. </li><li> Diluire i campioni di proteine ​​in tampone di caricamento SDS e caricare 20-40 mg di proteine ​​in gel 10% SDS-PAGE. </li><li> Eseguire gel SDS-PAGE a 20 mA per 1.5 ore. </li><li> Trasferimento proteine ​​di membrane PVDF in un sistema di trasferimento umida a 4 ° C per 3 ore a 85 V. Regolare il tempo di trasferimento secondo il peso molecolare delle proteine ​​bersaglio. </li><li> Membrana blocco in 5% di latte senza grassi per 30 minuti a 1 ora a temperatura ambiente. </li><li> Incubare la membrana with un anticorpo primario a 4 ° CO / N. La gamma di diluizione anticorpo primario è normalmente compresa tra 1: 200 e 1: 5.000, determinare la diluizione ottimale per ciascun anticorpo sperimentalmente. La diluizione di anticorpi utilizzati in questo protocollo può essere trovato in &quot;Tabella di reagenti e attrezzature specifiche&quot;. <ol><li> Per rilevare isoforme di splicing, utilizzare anticorpi specifici che riconoscono una particolare isoforma. In alternativa, utilizzare un anticorpo che riconosce l&#39;epitopo nella costitutiva esone regione codificante e rilevare le isoforme di splicing simultaneamente in base alle loro dimensioni diverse proteine. </li><li> Allo stesso tempo, monitorare EMT mediante immunoblotting per i marcatori di EMT. Utilizzare E-caderina, γ-catenina, e occludina come marcatori epiteliali, e fibronectina, N-caderina e vimentina come marcatori mesenchimali. </li></ol></li><li> Membrana Lavare tre volte con TBS-T (50 mM Tris, 150 mM NaCl, 0,05% Tween 20, pH 7.4) in un intervallo di 5 min. </li><li> Incubare la membrana con una formica secondario HRP-coniugatoiBody in una diluizione 1: 10.000 per 1 ora a RT. </li><li> Membrana Lavare tre volte con TBS-T in un intervallo di 5 min. </li><li> Visualizzare proteina con un sistema di rilevazione chemiluminescenza ed esporre la membrana di un film autoradiografia. </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recurrent+mutations+in+the+U2AF1+splicing+factor+in+myelodysplastic+syndromes.">Recurrent mutations in the U2AF1 splicing factor in myelodysplastic syndromes.</a> Nat Genet. 44, 53-57 (2012).</li><li>Papaemmanuil, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatic+SF3B1+mutation+in+myelodysplasia+with+ring+sideroblasts.">Somatic SF3B1 mutation in myelodysplasia with ring sideroblasts.</a> N Engl J Med. 365, 1384-1395 (2011).</li><li>Yoshida, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequent+pathway+mutations+of+splicing+machinery+in+myelodysplasia.">Frequent pathway mutations of splicing machinery in myelodysplasia.</a> Nature. 478, 64-69 (2011).</li><li>Mani, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epithelial-mesenchymal+transition+generates+cells+with+properties+of+stem+cells.">The epithelial-mesenchymal transition generates cells with properties of stem cells.</a> Cell. 133, 704-715 (2008).</li><li>Reinke, L. M., Xu, Y., Cheng, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Snail+represses+the+splicing+regulator+epithelial+splicing+regulatory+protein+1+to+promote+epithelial-mesenchymal+transition.">Snail represses the splicing regulator epithelial splicing regulatory protein 1 to promote epithelial-mesenchymal transition.</a> J Biol Chem. 287, 36435-36442 (2012).</li><li>Serini, G., Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+myofibroblast+activity+and+phenotypic+modulation.">Mechanisms of myofibroblast activity and phenotypic modulation.</a> Exp Cell Res. 250, 273-283 (1999).</li><li>Zavadil, J., Bottinger, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta+and+epithelial-to-mesenchymal+transitions.">TGF-beta and epithelial-to-mesenchymal transitions.</a> Oncogene. 24, 5764-5774 (2005).</li><li>Pfaffl, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+mathematical+model+for+relative+quantification+in+real-time+RT-PCR.">A new mathematical model for relative quantification in real-time RT-PCR.</a> Nucleic Acids Res. 29, e45 (2001).</li><li>Hellemans, J., Mortier, G., De Paepe, A., Speleman, F., Vandesompele, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=qBase+relative+quantification+framework+and+software+for+management+and+automated+analysis+of+real-time+quantitative+PCR+data.">qBase relative quantification framework and software for management and automated analysis of real-time quantitative PCR data.</a> Genome Biol. 8, R19 (2007).</li><li>Boutz, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+post-transcriptional+regulatory+switch+in+polypyrimidine+tract-binding+proteins+reprograms+alternative+splicing+in+developing+neurons.">A post-transcriptional regulatory switch in polypyrimidine tract-binding proteins reprograms alternative splicing in developing neurons.</a> Genes Dev. 21, 1636-1652 (2007).</li><li>Salomonis, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+regulates+mouse+embryonic+stem+cell+pluripotency+and+differentiation.">Alternative splicing regulates mouse embryonic stem cell pluripotency and differentiation.</a> Proc Natl Acad Sci USA. 107, 10514-10519 (2010).</li><li>Calarco, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vertebrate+nervous+system+alternative+splicing+and+development+by+an+SR-related+protein.">Regulation of vertebrate nervous system alternative splicing and development by an SR-related protein.</a> Cell. 138, 898-910 (2009).</li><li>Grabowski, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+takes+shape+during+neuronal+development.">Alternative splicing takes shape during neuronal development.</a> Curr Opin Genet Dev. 21, 388-394 (2011).</li><li>Seol, D. W., Billiar, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+caspase-9+variant+missing+the+catalytic+site+is+an+endogenous+inhibitor+of+apoptosis.">A caspase-9 variant missing the catalytic site is an endogenous inhibitor of apoptosis.</a> J Biol Chem. 274, 2072-2076 (1999).</li><li>Srinivasula, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+an+endogenous+dominant-negative+short+isoform+of+caspase-9+that+can+regulate+apoptosis.">Identification of an endogenous dominant-negative short isoform of caspase-9 that can regulate apoptosis.</a> Cancer Res. 59, 999-1002 (1999).</li><li>Akgul, C., Moulding, D. A., Edwards, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+splicing+of+Bcl-2-related+genes+functional+consequences+and+potential+therapeutic+applications.">Alternative splicing of Bcl-2-related genes functional consequences and potential therapeutic applications.</a> Cell Mol Life Sci. 61, 2189-2199 (2004).</li><li>Cooper, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+minigene+systems+to+dissect+alternative+splicing+elements.">Use of minigene systems to dissect alternative splicing elements.</a> Methods. 37, 331-340 (2005).</li><li>Cheng, C., Sharp, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+CD44+alternative+splicing+by+SRm160+and+its+potential+role+in+tumor+cell+invasion.">Regulation of CD44 alternative splicing by SRm160 and its potential role in tumor cell invasion.</a> Mol Cell Biol. 26, 362-370 (2006).</li></ol>

The authors declare that they have no competing financial interests.

La procedura qui descritta consente il rilevamento di splicing alternativo in un modello EMT inducibile. Come tali, le alterazioni dinamiche di espressione delle isoforme di splicing possono essere catturate nel corso tempo di EMT. Questo metodo ha vantaggi rispetto all&#39;uso di epithelial- diverso o linee di cellule mesenchimali-che rappresenta per il confronto di splicing alternativo, perché background genetici distinti da linee cellulari connessi alle Nazioni Unite potrebbero influenzare indebitamente splicing alt...

La transizione epitelio-mesenchimale (EMT) è un programma di sviluppo che guida la morfogenesi di organi e rimodellamento tissutale durante l&#39;embriogenesi. Quando anormalmente attivato, EMT promuove la metastasi del tumore e degli organi fibrosi 1,2. Studi convincenti hanno descritto l&#39;importanza della regolazione trascrizionale durante il processo di EMT, definito da diversi fattori di trascrizione, come il Twist, Lumaca, e ZEB, che reprimono l&#39;espressione della proteina di giunzione adherens E-caderina, con conseguente perdita di un cobble- pietra come morfologia epiteliale e il guadagno di un a forma di fuso fenotipo mesenchimale 3-8. Recenti studi attraverso l&#39;analisi dell&#39;intero genoma di RNA hanno rivelato che esiste un gruppo di geni i cui modelli di splicing sono associati sia con fenotipi epiteliali e mesenchimali 9,10. Il lavoro dal nostro laboratorio funzionalmente collegato splicing alternativo e EMT. Studiando la superficie cellulare molecola di adesione CD44, abbiamo dimostrato che il CD44 Alternative splicing è strettamente regolata durante EMT, e più importante, che CD44 giuntura isoforma passaggio causalmente contribuisce alla EMT 11. Splicing alternativo rappresenta un modello diffuso e conservato di regolazione genica, in quanto fino al 95% dei geni multi-esone umani sono splicing alternativo 12-14. Generando più prodotti proteici da un singolo gene, splicing alternativo costituisce un meccanismo essenziale per la diversità di proteine, aggiungendo un ulteriore livello di complessità al genoma umano. Come tale, disregolazione di splicing alternativo potrebbe potenzialmente portare a profondi effetti biologici che causano malattie umane. Infatti, aberrante splicing alternativo nelle malattie è stato documentato da oltre un decennio 15-25, compresi recenti scoperte che le mutazioni in geni che codificano per le macchine spliceosoma trovano comunemente nelle sindromi mielodisplastiche 26-28. Pertanto, lo sviluppo di metodi affidabili per la rilevazione di i splicing alternativosoforms è di grande importanza nello studio dei processi biologici diversi, tra cui EMT. Qui forniamo un protocollo per rilevare i cambiamenti nella splicing alternativo utilizzando un modello di EMT inducibile. I metodi per la progettazione di primer PCR e rilevazione isoforme di splicing sono adatte non solo per lo studio di splicing alternativo durante EMT, ma anche per lo studio di splicing alternativo in altri processi biologici. Indagare splicing alternativo durante EMT è indispensabile al fine di comprendere meglio i meccanismi di EMT e metastasi tumorali, facilitando così lo sviluppo di strategie efficaci per il trattamento di metastasi del cancro.

<table border="0" cellpadding="0" cellspacing="0" width="1167">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Name of the reagent</td>
			<td style="width:183px;">Company</td>
			<td style="width:123px;">Catalogue number</td>
			<td style="width:564px;">Comments (optional)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-hydroxytamoxifen</td>
			<td>Sigma</td>
			<td>H7904</td>
			<td>Make stock solution by dissolving in ethanol to 200&#956;M, and keep at -20&#8451; protected from light.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A. Total RNA Isolation kit</td>
			<td>Omega Bio-Tek</td>
			<td>R6731</td>
			<td>Total RNA isolation kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoScript Reverse Transcription System</td>
			<td>Promega</td>
			<td>A5001</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GoTaq qPCR Master Mix</td>
			<td>Promega</td>
			<td>A6002</td>
			<td>Reagent for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycler 480 Real-Time PCR System</td>
			<td>Roche</td>
			<td></td>
			<td>Equipment for qRT-PCR assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CD44 antibody</td>
			<td>R&#38;D Systems</td>
			<td>BBA10</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E-cadherin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>4065</td>
			<td>1:2500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#947;-catenin antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2309</td>
			<td>1:1000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">occludin antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-5562</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fibronectin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610077</td>
			<td>1:5000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N-cadherin antibody</td>
			<td>BD Transduction Laboratories</td>
			<td>610920</td>
			<td>1:2000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">vimentin antibody</td>
			<td>NeoMarkers</td>
			<td>MS-129-p1</td>
			<td>1:500 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GAPDH antibody</td>
			<td>Millipore Corporation</td>
			<td>MAB374</td>
			<td>1:10000 dilution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amasham ECL Western blotting detection reagent</td>
			<td>GE Health Life Science</td>
			<td>RPN2209</td>
			<td>Chemiluminescence system</td>
		</tr>
	</tbody>
</table>

detection of alternative splicing during epithelial-mesenchymal transition

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various physiological and pathological processes. Here we describe a strategy to detect alternative splicing during EMT using an inducible EMT model by expressing the transcription repressor Twist. EMT is monitored by changes in cell morphology, loss of E-cadherin localization at cell-cell junctions, and the switched expression of EMT markers, such as loss of epithelial markers E-cadherin and &#947;-catenin and gain of mesenchymal markers N-cadherin and vimentin. Using isoform-specific primer sets, the alternative splicing of interested mRNAs are analyzed by quantitative RT-PCR. The production of corresponding protein isoforms is validated by immunoblotting assays. The method of detecting splice isoforms described here is also suitable for the study of alternative splicing in other biological processes.

Le procedure sopra descritte forniscono un metodo affidabile per rilevare splicing alternativo durante EMT. Risultati rappresentativi di CD44 giunzione commutazione isoforma durante EMT Twist-indotta sono riportati di seguito come esempio. EMT Twist-indotta nelle cellule HMLE / Twist-ER è stato caratterizzato da una transizione da un ciottolo-pietra come fenotipo epiteliale ad un fenotipo fibroblastica allungata (Figura 2A), l&#39;assenza di marcatori epiteliali E-caderina,...

Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Rilevamento di splicing alternativo Durante epiteliale-mesenchimale transizione

Alternative splicing plays a critical role in the epithelial-mesenchymal transition (EMT), an essential cellular program that occurs in various ...

detection-alternative-splicing-during-epithelial-mesenchymal

Research

JoVE Journal

Biology

12.8K Views.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Video: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Light/dark Transition Test for Mice

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, however, can be used to delete or manipulate a specific gene in mice. The influence of a given gene on a specific behavior can then be determined by conducting behavioral analyses of the mutant mice. As a test for behavioral phenotyping of mutant mice, the light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. The test is based on the natural aversion of mice to brightly illuminated areas and on their spontaneous exploratory behavior in novel environments. The test is sensitive to anxiolytic drug treatment. The apparatus consists of a dark chamber and a brightly illuminated chamber. Mice are allowed to move freely between the two chambers. The number of entries into the bright chamber and the duration of time spent there are indices of bright-space anxiety in mice. To obtain phenotyping results of a strain of mutant mice that can be readily reproduced and compared with those of other mutants, the behavioral test methods should be as identical as possible between laboratories. The procedural differences that exist between laboratories, however, make it difficult to replicate or compare the results among laboratories. Here, we present our protocol for the light/dark transition test as a movie so that the details of the protocol can be demonstrated. In our laboratory, we have assessed more than 60 strains of mutant mice using the protocol shown in the movie. Those data will be disclosed as a part of a public database that we are now constructing.

Visualization of the protocol will facilitate understanding of the details of the entire experimental procedure, allowing for standardization of the protocols used across laboratories and comparisons of the behavioral phenotypes of various strains of mutant mice assessed using this test.

The light/dark transition test is one of the most widely used tests to measure anxiety-like behavior in mice. Here, we present a movie that shows detailed procedures on how we conduct the test.

Luce / buio test di transizione per i Topi

Although all of the mouse genome sequences have been determined, we do not yet know the functions of most of these genes. Gene-targeting techniques, ...

Ricerca

Biologia

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

Rilevamento di proteine ​​Ubiquitinazione

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Optimized PCR-based Detection of Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. Although mycoplasma do not usually kill contaminated cells, they are difficult to detect and can cause a variety of effects on cultured cells, including altered metabolism, slowed proliferation and chromosomal aberrations. In short, mycoplasma contamination compromises the value of those cell lines in providing accurate data for life science research.
The sources of mycoplasma contamination in the laboratory are very challenging to completely control. As certain mycoplasma species are found on human skin, they can be introduced through poor aseptic technique. Additionally, they can come from contaminated supplements such as fetal bovine serum, and most importantly from other contaminated cell cultures. Once mycoplasma contaminates a culture, it can quickly spread to contaminate other areas of the lab. Strict adherence to good laboratory practices such as good aseptic technique are key, and routine testing for mycoplasma is highly recommended for successful control of mycoplasma contamination.
PCR-based detection of mycoplasma has become a very popular method for routine cell line maintenance. PCR-based detection methods are highly sensitive and can provide rapid results, which allows researchers to respond quickly to isolate and eliminate contamination once it is detected in comparison to the time required using microbiological techniques. The LookOut Mycoplasma PCR Detection Kit is highly sensitive, with a detection limit of only 2 genomes per &mu;l. Taking advantage of the highly specific JumpStart Taq DNA Polymerase and a proprietary primer design, false positives are greatly reduced. The convenient 8-tube format, strips pre-coated with dNTPs, and associated primers helps increase the throughput to meet the needs of customers with larger collections of cell lines.
Given the extreme sensitivity of the kit, great care must be taken to prevent inadvertent contamination of samples and reagents. The step-by-step protocol we demonstrate highlights the precautions and practices required for reliable mycoplasma detection. We also show and discuss typical results and their interpretation. Our goal is to ensure the success of researchers using the LookOut Mycoplasma PCR Detection Kit.

The LookOut Mycoplasma PCR Detection Kit utilizes the polymerase chain reaction (PCR), which is established as the method of choice for highest sensitivity in the detection of Mycoplasma, Acholeplasma, and Ureaplasma contamination in cell cultures and other cell culture derived biologicals.

Ottimizzati basati sulla PCR Rilevazione di Mycoplasma

The maintenance of contamination-free cell lines is essential to cell-based research. Among the biggest contaminant concerns are mycoplasma contamination. ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

Multi-parametro di misura della apertura dei pori di transizione di permeabilità nel Cuore mouse mitocondri isolati

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an attractive model system to study organ development and tissue-specific signaling. Although hair follicle development is genetically tractable, fast and reproducible analysis of factors essential for this process remains a challenge. Here we describe a procedure to generate targeted overexpression or shRNA-mediated knockdown of factors using lentivirus in a tissue-specific manner. Using a modified version of a hair regeneration model 5, 6, 11, we can achieve robust gain- or loss-of-function analysis in primary mouse keratinocytes or dermal cells to facilitate study of epithelial-mesenchymal signaling pathways that lead to hair follicle morphogenesis. We describe how to isolate fresh primary mouse keratinocytes and dermal cells, which contain dermal papilla cells and their precursors, deliver lentivirus containing either shRNA or cDNA to one of the cell populations, and combine the cells to generate fully formed hair follicles on the backs of nude mice. This approach allows analysis of tissue-specific factors required to generate hair follicles within three weeks and provides a fast and convenient companion to existing genetic models.

Tissue-specific analysis of a hair follicle regeneration model using lentivirus to mediate gain- or loss-of-function.

Rapida analisi genetica di epitelio-mesenchimale segnalazione durante la rigenerazione dei capelli

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Induzione e Analisi dei epiteliale a Mesenchymal Transizione

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Le colture per pluripotenti umane alternative staminali produzione di celle, manutenzione e analisi genetica

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Methods to Discover Alternative Promoter Usage and Transcriptional Regulation of Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually untranslated &#8212; first exons. Bcrp1 (Abcg2), the murine orthologue of the ABC transporter Breast Cancer Resistance Protein (BCRP, ABCG2), has at least four alternative promoters that are designated by the corresponding four alternative first exons produced: E1U, E1A, E1B, and E1C. Herein, in-silico protocols are presented to predict alternative promoter usage for Bcrp1. Furthermore, reporter assay methods are described to produce reporter constructs for alternative promoters and to determine the functionality of putative promoters upstream of the alternative first exons that are identified.

With the murine ABC transporter Bcrp1 (Abcg2) as an example, in-silico protocols are presented to detect alternative promoter usage in genes expressed in mouse tissues, and to evaluate the functionality of the alternative promoters identified using reporter assays.

I metodi per scoprire alternative Uso Promotore e regolazione trascrizionale di Murine Bcrp1

Gene expression in different tissues is often controlled by alternative promoters that result in the synthesis of mRNA with unique &#8212; usually ...

An Alternative Approach for Sample Preparation with Low Cell Number for TEM Analysis

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, especially cells in suspension such as hematopoietic stem cells (HSCs), remains limited due to the requirement of a high cell number during sample preparation. There are a few cytospin or monolayer approaches for TEM analysis from scarce samples, but these approaches fail to get significant quantitative data from the limited number of cells. Here, an alternative and novel approach for sample preparation in TEM studies is described for rare cell populations that enables quantitative analysis.
A relatively low cell number, i.e., 10,000 HSCs, was successfully used for TEM analysis compared to the millions of cells typically used for TEM studies. In particular, Evans blue staining was performed after paraformaldehyde-glutaraldehyde (PFA-GA) fixation to visualize the tiny cell pellet, which facilitated embedding in agarose. Clusters of numerous cells were observed in ultra-thin sections. The cells had a well preserved morphology, and the ultra-structural details of the Golgi complex and several mitochondria were visible. This efficient, easy and reproducible protocol allows sample preparation from a low cell number and can be used for qualitative and quantitative TEM analysis on rare cell populations from limited biological samples.

Here, we present a protocol to prepare samples with low cell numbers for transmission electron microscopy (TEM) analysis.

Un approccio alternativo per la preparazione del campione con un basso numero di cellule per l&#39;analisi TEM

Transmission electron microscopy (TEM) provides details of the cellular organization and ultrastructure. However, TEM analysis of rare cell populations, ...

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with organ fibrosis. In these conditions, differentiated endothelial cells acquire mesenchymal-like phenotypes. This process is called endothelial-mesenchymal transition (EndMT) and is characterized by downregulation of endothelial markers, upregulation of mesenchymal markers, and morphological changes. EndMT is induced by several signaling pathways, including transforming growth factor (TGF)-&#946;, Wnt, and Notch, and regulated by molecular mechanisms similar to those of epithelial-mesenchymal transition (EMT) important for gastrulation, tissue fibrosis, and cancer metastasis. Understanding the mechanisms of EndMT is important to develop diagnostic and therapeutic approaches targeting EndMT. Robust induction of EndMT in vitro is useful to characterize common gene expression signatures, identify druggable molecular mechanisms, and screen for modulators of EndMT. Here, we describe an in vitro method for induction of EndMT. MS-1 mouse pancreatic microvascular endothelial cells undergo EndMT after prolonged exposure to TGF-&#946; and show upregulation of mesenchymal markers and morphological changes as well as induction of multiple inflammatory chemokines and cytokines. Methods for the analysis of microRNA (miRNA) modulation are also included. These methods provide a platform to investigate mechanisms underlying EndMT and the contribution of miRNAs to EndMT.

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&amp;#946; Signaling

A protocol for in vitro induction of endothelial-mesenchymal transition (EndMT), which is useful for investigating cellular signaling pathways involved in EndMT, is described. In this experimental model, EndMT is induced by treatment with TGF-&#946; in MS-1 endothelial cells.

Analisi molecolare di transizione endoteliali mesenchimali indotta dalla trasformazione di segnalazione di fattore di crescita-β

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...