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. Zellkultur von EMT Induktions Hinweis: EMT kann durch die Behandlung von TGFß oder ektopische Expression von Transkriptionsfaktoren Twist, Schnecke, oder ZEB1 / 2 in Epithelzellen induziert werden. In diesem Protokoll beschrieben ist eine induzierbare EMT-System über die Expression des Twist-ER-Fusionsprotein in verewigt menschlichen Brust Epithelzellen (HMLE / Twist-ER, ein Geschenk von Dr. J Yang, UCSD) 9,11,29. Auf 4-Hydroxytamoxifen (TAM) Behandlung transloziert das Fusionsprotein Twist-ER in den Kern, um die Transkription und führt zu einer vollständigen EMT Übergang in 12-14 Tagen 9,11,29 fahren. Zusätzliche EMT induzierbare Systeme, wie HMLE / Snail-ER, HMLE / TGFß und MCF10A / TGFß, finden Sie in unseren früheren Veröffentlichungen 11,30 gefunden werden. <ol><li> Aufrechterhaltung HMLE / Twist-ER Zellen in serumfreiem Brustepithelzellen Zellwachstumsmedium (MEGM) in einem 37 ° C Inkubator mit 5% CO 2. Durchgang die Zellen alle 2-3 Tage, wenn sie 80% Konfluenz erreichen. Zellen mit 0,15% Trypsin bei 37 ° C für 5-10 min, und anschließend mit Trypsin zu inaktivieren, 5% Kälberserum / DMEM. Spin-down-Zellen und resuspendieren sie in MEGM. Plattenzellen in einer neuen Gewebekulturschale in einem Verhältnis von 1 bis 4 ist. </li><li> TAM in Ethanol auflösen, um eine 200 uM Lösung. TAM schützen vor Licht und bei -20 ° C. Vor Gebrauch frisch TAM in MEGM Medien Hinzufügen bei einer 1: 10.000 Verdünnung auf eine Endkonzentration von 20 nM TAM machen. </li><li> Für die Induktion der EMT, Platte 1,6 x 10 6 HMLE / Twist-ER-Zellen in einer 10 cm Gewebekulturschale in zweifacher Ausfertigung. Kulturzellen mit 20 nM TAM haltigen MEGM Medien. </li><li> Platte 1,6 x 10 6 HMLE / Twist-ER-Zellen in einer 10 cm-Schale in zweifacher Ausfertigung und Behandlung Zellen mit 0,01% Ethanol als nicht-TAM-behandelten Kontroll. </li><li> Zwei Tage nach der Inkubation, machen Sie Fotos unter einem Lichtmikroskop bei 10-facher Vergrößerung zu morphologischen Veränderungen der Zellen zu erfassen. </li><li> Saugen Sie das Medium aus einem Gericht der Kontrolle oder TAM-behandeltZellen. Waschen Sie die Zellen mit kaltem PBS und Ernte von Zellen Verschrottung Hälfte der Platte in der RNA-Lyse-Puffer für RNA-Isolierung und die andere Hälfte in RIPA-Puffer für die Proteinanalyse. </li><li> Passage-Zellen aus der Steuerung oder TAM-behandelten Gerichten, die von Re-Plattierung 1,6 x 10 6 Zellen in einer 10 cm-Schale in zweifacher Ausfertigung. Kontinuierlich behandeln die Zellen mit 20 nM TAM oder Fahrzeug. </li><li> Wiederholen Sie die Schritte 5 bis 7 einmal jeden zweiten Tag bis zum 14. Tag, zu welcher Zeit, TAM-behandelten Twist-ER-Zellen zeigen eine spindelförmige Morphologie mesenchymalen, während Vehikel-behandelten Kontrollzellen pflegen die Kopfsteinpflaster-wie epitheliale Morphologie. </li></ol> 2. Charakterisierung EMT Hinweis: Ein Abschluss der EMT wird angezeigt durch: (1) Handy morphologische Veränderungen von einem pflastersteinartigen epithelialen Morphologie zu einer spindelförmigen Fibroblasten-ähnliche Aussehen; (2) Der Verlust der E-Cadherin-Lokalisation an Zell-Zell-Verbindungen; und (3) Der Schalt Expression von EMT-Marker, die durch den Verlust definiertepithelialen Marker und Verstärkung von mesenchymalen Marker. Zelle morphologische Veränderungen durch Lichtmikroskop bei 10-facher Vergrößerung im Zeitverlauf der Induktion EMT erfasst, in Abschnitt 1. Immuno-Fluoreszenz-Detektion von E-Cadherin bei Zellen-Übergängen beschrieben, ist in diesem Abschnitt beschrieben. Die Expression von EMT-Marker wird durch Immunoblotting nachgewiesen. Gemeinsamen epithelialen Marker E-Cadherin, γ-Catenin und Occludin und mesenchymalen Marker umfassen Fibronektin, N-Cadherin und Vimentin. Allgemeine Vorgänge der Immunoblot werden in Abschnitt 4 beschrieben. <ol><li> Am Tag 12 der Behandlung TAM, Samen 1 x 10 5 Zellen auf einer 12 mm Glaskreisdeckglas in eine Vertiefung einer 24-Well-Platte gelegt. </li><li> 48 Stunden nach dem Aussäen absaugen mittel-und Wasch Zellen mit vorgewärmter PBS. </li><li> Fix Zellen mit vorgewärmten 4% Paraformaldehyd für 15 min. </li><li> Mit PBS waschen Zellen dreimal. </li><li> Zellen mit 0,2% Triton X-100 in PBS Inkubieren für 15min bei RT. </li><li> Mit PBS waschen Zellen dreimal. </li><li> Zellen in 5% BSA / PBS-Inkubation für 1 h bei RT zu blockieren nichtspezifische Bindung. </li><li> Zellen, die mit dem primären Antikörper E-Cadherin Inkubieren bei 1:50 Verdünnung in 1% BSA / PBS in einer befeuchteten Kammer O / N bei 4 ° C ist. HINWEIS: Der Bereich für primäre Antikörper Verdünnung ist normalerweise zwischen 1:50 und 1: 200, und die optimale Verdünnung für jeden Antikörper sollte experimentell bestimmt werden. </li><li> Dekantiert man die primären Antikörper und waschen Sie die Zellen dreimal mit PBS. </li><li> Zellen mit fluoreszenzmarkierten sekundären Antikörper inkubieren in einer 1: 500-Verdünnung für 1 h bei RT. Von diesem Schritt auf, schützen die Zellen vor Licht. </li><li> Dekantiert man die sekundären Antikörper und dreimal mit PBS. </li><li> Fleck Zellen mit 0,1-1 g / ml DAPI oder Hoechst für 10 min. </li><li> Spülen Zellen dreimal mit PBS. </li><li> Berg Deckgläschen mit einem Tropfen Eindeckmedium und Deckgläschen Dichtung mit Nagellack. </li><li> Beachten Zellen und nehmenBilder unter einem Fluoreszenzmikroskop 63X. </li></ol> Färbung mit zusätzlichen Antikörpern kann durchgeführt werden, um die EMT-Phänotyp zu bestätigen. Zum Beispiel können N-Cadherin und α-Aktin des glatten Muskels bei einer mesenchymalen Phänotyp 29,31,32 gefärbt werden. Reorganisation des Zytoskeletts Struktur kann durch Anfärben der Zellen mit Phalloidin für F-Actin-11 überwacht werden. Darüber hinaus können Assays auf Zellbeweglichkeit und Zelltod Widerstand durchgeführt werden, um die Eigenschaften des EMT 11 zu prüfen. 3. Nachweis der Splice-Isoformen Mit qRT-PCR-Assays <ol><li> Primer-Design HINWEIS: Primerpaare sollten sorgfältig geplant werden, um unterschiedliche Spleiß-Isoformen zu verstärken. Exon-Skipping, das am häufigsten beobachteten alternatives Spleißen Fall wird hier als Beispiel für die Strategie der Primer-Design veranschaulichen. 1A zeigt einen Vier-Exon-pre-mRNA, wobei das dritte Exon als variable Exon. Einbeziehung von Exon 3 Ergebnisse indie Herstellung eines mehr Spleiß-Isoform, wobei das Exon 3 erzeugt einen kürzeren Spleiß-Isoform. Primer müssen entwickelt, um die Exon-3-Isoform enthalten und Exon-3-Isoform unterscheiden sprungen, und die Gesamt Transkript dieser mRNA zu detektieren. <ol><li> Für den Nachweis von Exons Aufnahme sorgfältig Design eine Vorwärtsprimer innerhalb des variablen Exon 3 und einem Reverse-Primer in der Nähe konstitutive Exon 4, so dass für die spezifische Amplifikation der variablen Exon-Isoform enthalten. Achten Sie darauf, sowohl vorwärts als auch entwerfen und Reverse-Primer innerhalb der gleichen variable Exon, um PCR-Produkte aus genomischer DNA oder pre-mRNA verstärkt zu vermeiden. </li><li> Zur spezifischen Amplifizierung Exon-Skipping-Ereignisse, Design einer der Primer überspannt den Übergang des konstitutiven Exon 2 und Exon 4, die auf andere Weise zerstört würde, wenn variable Exon 3 enthalten sein. Entwerfen der andere Primer in dem konstitutiven Exon 4. Daher werden PCR-Produkte erzeugt, nur wenn der variable Exon 3 excludEd und die konstitutive Exon 2 und Exon 4 werden anschließend miteinander verbunden. </li><li> Um die Gesamt Transkripte des Gens zu erkennen, entwerfen die Primer innerhalb von zwei konstitutiven Exons 1 und 2. So werden PCR-Produkte aus allen Isoformen verstärkt. </li><li> Sicherzustellen, dass die Schmelztemperatur (Tm) für alle Primer in etwa 55 ° C, wobei die Länge jedes Primers etwa 18-22 Nukleotide, und die Größe der PCR-Produkte liegt zwischen 80 und 150 Basenpaaren. </li></ol></li><li> RNA-Extraktion: Auszug RNA mit einer Gesamt-RNA Isolation Kit (siehe Tabelle der Materialien). <ol><li> Sammeln Zellen in 350 ul RNA Lyse-Puffer. </li><li> Fügen Sie 350 ul 70% Ethanol zum Lysat und gründlich mischen. </li><li> Übertragung der Probe auf die Säule zur Reinigung von RNA. </li><li> Zentrifuge bei 10.000 g für 1 min und Durchfluss verwerfen. </li><li> Einmal mit 500 ul Waschpuffer I RNA und RNA zweimal mit Waschpuffer II Die Säule. </li><li> Reste von RNA-WaschpufferII von der Säule durch Zentrifugation bei maximaler Geschwindigkeit für 2 Minuten. </li><li> Übertragen die Säule in ein neues 1,5-ml-Röhrchen werden 50 ul Nuklease-freies Wasser in der Mitte des Säulenmatrix und Eluieren RNA durch Zentrifugation bei maximaler Geschwindigkeit. </li></ol></li><li> Synthese von Erststrang-cDNA <ol><li> Bestimmung der Konzentration und Qualität der RNA durch UV-Absorptionsmessung. HINWEIS: Gute Qualität RNA sollte eine 260 nm / 280 nm Absorptionsverhältnis von etwa 2,0. </li><li> Der reverse Transkriptase (RT)-Reaktionen, die 250 ng Gesamt-RNA, 0,05 ug Zufalls-Hexamer-Primer, 50 pmol MgCl2, 10 pmol dNTP, 2 ul 5 · GoScript Puffer und 1 ul RT-Enzym in einem Endvolumen von 20 ul enthalten eingestellt. </li><li> Führen RT-Reaktionen bei 42 ° C für 1 h, gefolgt von einer Inkubation bei 70 ° C für 5 min, um die RT-Enzym zu inaktivieren. </li></ol></li><li> Echtzeit-PCR <ol><li> Bis 20 ul Reaktionen, die von 10 ul SYBR Green Master Mix bestehen eingestellt,0,1-1,0 ul cDNA-Vorlage, und 5 pmol Vorwärts und Rückwärts-Primer. </li><li> Führen Echtzeit-PCR mit 40 Zyklen der folgenden Schritte: 95 ° C Denaturierung für 10 Sekunden bei 58 ° C Annealing für 10 sec und 60 ° C Extension für 30 Sekunden. Führen PCR-Reaktionen in dreifacher Ausfertigung. </li><li> Überprüfen Dissoziationskurven und stellen Sie sicher, dass ein einzelner Peak ist für jedes Primerpaar beobachtet. </li><li> Erhalten die Werte Quantifizierung Zyklus (CQ) durch die Berechnung der zweiten Ableitung Werte der Verstärkungskurven. </li><li> Berechnen der relativen Menge (RQ) des Ziel-mRNAs in induzierten Proben im Vergleich zu der nicht-induzierten Probe unter Verwendung des &#39;Delta-Delta-Cq (ΔΔCq) &quot;-Methode, wie durch die folgende Formel gezeigt. Verwenden Housekeeping-Gene, wie GAPDH oder TBP, als Referenz. </li></ol></li></ol><img alt="Gleichung 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Um genauer zu berechnen relativen Werte von Spleiß-Isoformen, ter von mehreren Referenzgenen verwenden sollten berücksichtigt werden. Die Ergebnisse können unter Verwendung eines von Pfaffl et al 33,34 beschriebenen modifizierten Absolutwert Quantifizierungsmethode analysiert werden. 4. Prüfung der Splice-Isoformen auf Proteinebene <ol><li> Sammeln Zellen in 100 ul eiskaltem RIPA-Puffer. </li><li> Stellen Lysate auf Eis für etwa 10 min. </li><li> Zentrifuge bei 14.000 × g für 10 min bei 4 ° C und Sammeln des Überstandes. </li><li> Messung der Proteinkonzentration durch Bradford-Assays. </li><li> Verdünnte Proteinproben in SDS-Beladungspuffer geladen 20-40 ug Proteine ​​in 10% SDS-PAGE-Gelen. </li><li> Führen SDS-PAGE-Gelen bei 20 mA für 1,5 Stunden. </li><li> Transfer-Proteine ​​auf PVDF-Membranen in einer Nasstransfersystem bei 4 ° C für 3 h bei 85 V. Einstellen der Übertragungszeit nach dem Molekulargewicht des Zielproteins. </li><li> Block Membran in 5% fettfreier Milch 30 min bis 1 h bei RT. </li><li> Inkubieren Membran witen einen primären Antikörper bei 4 ° CO / N. Der Bereich für die Primärantikörperverdünnung in der Regel zwischen 1: 200 und 1: 5.000, bestimmen die optimale Verdünnung für jeden Antikörper experimentell. Die Verdünnung der Antikörper in diesem Protokoll verwendet werden, können in &quot;Tabelle der spezifischen Reagenzien und Geräte&quot; zu finden. <ol><li> Um Spleiß-Isoformen erkennt, verwenden Sie spezifische Antikörper, die eine bestimmte Isoform zu erkennen. Alternativ können einen Antikörper, der das Epitop im konstitutiven Exon codierenden Region erkennt und erfassen Spleiß-Isoformen gleichzeitig aufgrund ihrer unterschiedlichen Proteingrößen. </li><li> Zur gleichen Zeit, zu überwachen EMT durch Immunoblotting EMT-Marker. Sie können den E-Cadherin, γ-Catenin und Occludin als epithelialer Marker und Fibronektin, N-Cadherin und Vimentin als mesenchymalen Marker. </li></ol></li><li> Waschen der Membran dreimal mit TBS-T (50 mM Tris, 150 mM NaCl, 0,05% Tween 20, pH 7,4) in einem 5-Minuten-Intervall. </li><li> Inkubieren Membran mit einem HRP-konjugierten sekundären Ameisenibody in einer 1: 10.000-Verdünnung für 1 h bei RT. </li><li> Waschen der Membran dreimal mit TBS-T bei einer 5-min-Intervall. </li><li> Visualisieren Protein mit einem Chemilumineszenz-Detektionssystem und setzen die Membran auf eine Autoradiographiefilm. </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.

Das hier beschriebene Verfahren ermöglicht den Nachweis des alternativen Spleißens in einem induzierbaren EMT-Modell. Als solche dynamische Veränderungen der Spleiß-Isoform-Expression im gesamten zeitlichen Verlauf der EMT erfasst werden. Diese Methode hat Vorteile gegenüber der Verwendung von unterschiedlichen epithelial- oder mesenchymalen-Zelllinien, die für den Vergleich von alternativen Spleißen, weil verschiedene genetische Hintergründe aus un-verwandten Zelllinien konnte mäßig beeinflussen alternative S...

Die epitheliale-mesenchymale Transition (EMT) ist ein Entwicklungsprogramm, das Organ Morphogenese und Gewebeumbau während der Embryogenese steuert. Wenn abnorm aktiviert, fördert die EMT Tumormetastasierung und Organfibrose 1,2. Zwingende Studien haben die Bedeutung der transkriptionellen Regulierung während des Prozesses der EMT beschrieben, durch mehrere Transkriptionsfaktoren, wie Verdrehen, Schnecke, und ZEB, die die Expression des Zonula-junction-Protein E-Cadherin-Repression, was zum Verlust eines Pflaster definiert Stein wie epitheliale Morphologie und Verstärkung eines spindelförmigen mesenchymalen Phänotyp 3-8. Jüngste Studien durch genomweiten Analyse von RNAs aufgedeckt, daß es eine Gruppe von Genen, deren Klebemuster entweder mit epithelialen oder mesenchymalen Phänotypen 9,10 zugeordnet. Arbeiten aus unserem Labor alternatives Spleißen und EMT funktionell verbunden. Durch das Studium der Zelloberfläche Adhäsionsmolekül CD44 wir gezeigt, dass CD44 alterntiven Spleißen dicht bei EMT geregelt, und noch wichtiger, dass CD44 Spleiß-Isoform Schalt kausal trägt EMT 11. Alternatives Spleißen ist ein weit verbreitet und konservierten Modell der Genregulation, wie bis zu 95% der menschlichen Multi-Exon-Gene sind alternativ gespleißte 12-14. Durch die Erzeugung von mehreren Protein-Produkte von einem einzigen Gen, stellt alternative Spleißen einen wesentlichen Mechanismus für die Proteinvielfalt, indem eine weitere Schicht von Komplexität des menschlichen Genoms. Als solche könnte eine Fehlregulation von alternativem Spleißen möglicherweise tiefgreifende biologische Wirkungen verursachen Erkrankungen des Menschen führen. Tatsächlich hat abweichende alternative Spleißen in Krankheiten, für mehr als ein Jahrzehnt 15-25 dokumentiert, einschließlich der jüngsten Erkenntnisse, die Mutationen in Genen die Spleißosom Maschinen codieren, werden häufig in myelodysplastischen Syndromen 26-28 gefunden. Daher ist die Entwicklung zuverlässiger Verfahren zum Nachweis von alternativ gespleißten isoforms ist von großer Bedeutung in der Erforschung der vielfältigen biologischen Prozesse, einschließlich EMT. Hier bieten wir ein Protokoll, um Veränderungen in alternative Spleißen unter Verwendung eines induzierbaren EMT-Modell zu erkennen. Die Verfahren zum Entwerfen von PCR-Primern und das Nachweisen Spleiß-Isoformen sind nicht nur für die Untersuchung von alternativem Spleißen während EMT, sondern auch zur Untersuchung des alternativen Spleißens in anderen biologischen Prozessen. Untersuchung alternatives Spleißen während EMT ist zwingend notwendig, um ein besseres Verständnis der Mechanismen der EMT und Metastasierung von Tumoren, so die Entwicklung von Strategien, um die Krebsmetastasen zu behandeln.

<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.

Die oben beschriebenen Verfahren stellen eine robuste Methode zur alternatives Spleißen während EMT erfassen. Repräsentative Ergebnisse der CD44 splice Isoform Schalt bei Twist-induzierte EMT sind nachfolgend als Beispiel angegeben. Twist-induzierte EMT in HMLE / Twist-ER-Zellen wurde durch einen Übergang von einem Kopfstein wie epitheliale Phänotyp zu einem länglichen Fibroblasten-Phänotyp (2A), das Fehlen von epithelialen Marker E-Cadherin, γ-Catenin und Occludin u...

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.

Nachweis von Alternative Splicing Während Epithelial-mesenchymale Transition

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 Aufrufe.  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.

Serie: 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.

Hell / Dunkel Transition Test für Mäuse

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, ...

Forschung

Biologie

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.

Detektion von Protein Ubiquitination

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.

Optimierte PCR-basierten Nachweis von 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-Parameter-Messung der Durchlässigkeit Transition Pore Opening in isolierten Maus-Mitochondrien

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.

Schnelle Genetische Analyse von epithelial-mesenchymale Signaling Während Haarregeneration

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.

Induktion und Analyse von epithelialen zu mesenchymalen Transition

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.

Alternative Kulturen für menschlichen pluripotenten Stammzellen Produktion, Wartung und Genetic Analysis

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.

Methoden Alternative Promoter Verwendung und Transkriptions-Verordnung von Murine Bcrp1 zu entdecken

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.

Ein alternativer Ansatz für die Probenvorbereitung mit geringer Zellzahl für die TEM-Analyse

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.

Molekulare Analyse der endothelialen Mesenchymale Transition induziert durch Transforming Growth Factor-β-Signalisierung

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