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. cultivo celular de EMT Inducción Nota: EMT puede ser inducida por tratamiento de TGF o la expresión ectópica de factores de transcripción Twist, Snail, o ZEB1 / 2 en las células epiteliales. Se describe en este protocolo es un sistema inducible EMT través de la expresión de la proteína de fusión Twist-ER en las células mamarias humanas inmortalizadas epiteliales (HMLE / Twist-ER, un regalo del Dr. J Yang, UCSD) 9,11,29. Tras 4-hidroxitamoxifeno tratamiento (TAM), la proteína de fusión Twist-ER se transloca al núcleo para conducir la transcripción y los resultados en una transición EMT completa en 12-14 días 9,11,29. Sistemas inducibles EMT adicionales, como HMLE / Caracol-ER, HMLE / TGF, y MCF10A / TGF, se pueden encontrar en nuestras publicaciones anteriores 11,30. <ol><li> Mantener células HMLE / desenroscables ER en mamaria medio de crecimiento de células epiteliales sin suero (MEGM) en una incubadora a 37 ° con 5% de CO 2. Pasaje de las células cada 2-3 días cuando llegan a 80% de confluencia. Se incuban las células con 0,15% de tripsina a 37 ° C durante 5-10 min, y después inactivar la tripsina con suero de ternera 5% / DMEM. Centrifugar las células y resuspender en MEGM. Células de la placa en una nueva placa de cultivo tisular en una proporción de 1 a 4. </li><li> Disolver TAM en etanol para preparar una solución 200 mM. Proteja TAM de la luz y almacenar a -20 ° C. Antes de su uso, recién añadir TAM en medios MEGM a una dilución de 1: 10.000 para hacer una concentración final de 20 nM TAM. </li><li> Para la inducción de la EMT, placa células 1,6 x 10 6 HMLE / desenroscables ER en una placa de 10 cm de cultivo de tejidos por duplicado. Células de cultivo con medios 20 nM TAM-que contiene MEGM. </li><li> Placa células 1,6 x 10 6 HMLE / desenroscables ER en una placa de 10 cm por duplicado y el tratamiento de las células con etanol 0,01% como control no tratada con TAM. </li><li> Dos días después de la incubación, tomar fotografías bajo un microscopio de luz a un aumento de 10X para registrar los cambios morfológicos de las células. </li><li> Aspirar el medio de un plato de control o tratados con TAMcélulas. Lavar las células con PBS frío, y las células de la cosecha mediante el desguace de un medio de la placa en tampón de lisis de ARN para el aislamiento de ARN y la otra mitad en tampón RIPA para el análisis de proteínas. </li><li> Células pasaje del control o platos tratados con TAM en 1,6 x 10 6 células re-enchapado en una placa de 10 cm por duplicado. Continuamente tratar las células con 20 nM de TAM o vehículo. </li><li> Repita los pasos 5 a 7 una vez cada dos días hasta el día 14, momento en el cual, las células Twist-ER tratados con TAM muestran una morfología mesenquimal fusiforme, mientras que las células de control tratados con vehículo mantienen la morfología epitelial-adoquines similares. </li></ol> 2. Caracterización de EMT Nota: A la finalización de la EMT se indica mediante: (1) celulares cambios morfológicos de una morfología epitelial-adoquines como éste adopte un aspecto fibroblástico en forma de huso; (2) Pérdida de localización E-cadherina en las uniones célula-célula; y (3) La expresión de los marcadores de EMT de conmutación, que se define por la pérdidade marcadores epiteliales y ganancia de marcadores mesenquimales. Cambios morfológicos celulares son capturados por microscopio de luz a un aumento de 10X durante el curso del tiempo de la inducción de EMT, descrito en la Sección 1.-Immuno fluorescencia detección de E-cadherina en células uniones se describe en esta sección. La expresión de marcadores de EMT se detecta por inmunotransferencia. Marcadores epiteliales comunes son la E-cadherina, γ-catenina, y ocludina y marcadores mesenquimales incluyen fibronectina, N-cadherina y vimentina. Procedimientos generales de inmunotransferencia se describen en la Sección 4. <ol><li> En el día 12 del tratamiento TAM, semilla de 1 x 10 5 células en un 12 mm de vidrio cubreobjetos circular colocado en un pocillo de una placa de 24 pocillos. </li><li> 48 horas después de la siembra de células, medio aspirado y lavado con PBS precalentado. </li><li> Fijar las células con pre-calentado paraformaldehído al 4% durante 15 min. </li><li> Lavar las células tres veces con PBS. </li><li> Se incuban las células con 0,2% de Triton X-100 en PBS durante 15min a TA. </li><li> Lavar las células tres veces con PBS. </li><li> Se incuban las células en 5% de BSA / PBS durante 1 hora a TA para bloquear la unión no específica. </li><li> Se incuban las células con el anticuerpo primario E-cadherina en una dilución 1:50 en 1% de BSA / PBS en una cámara humidificada O / N a 4 ° C. NOTA: El intervalo de dilución de anticuerpo primario es normalmente entre 1:50 y 1: 200, y la dilución óptima para cada anticuerpo debe ser determinado experimentalmente. </li><li> Decantar el anticuerpo primario y lavar las células tres veces con PBS. </li><li> Se incuban las células con anticuerpo secundario marcado con fluorescencia en una dilución 1: 500 durante 1 hora a RT. Desde este paso en, proteger las células de la luz. </li><li> Decantar el anticuerpo secundario y lavar tres veces con PBS. </li><li> Las células se tiñen con 0,1-1 g / ml DAPI o Hoechst durante 10 min. </li><li> Enjuagar las células con PBS tres veces. </li><li> Monte cubreobjetos con una gota de medio de montaje y cubreobjetos sello con esmalte de uñas. </li><li> Observar las células y tomarimages bajo un microscopio de fluorescencia 63X. </li></ol> La tinción con anticuerpos adicionales se puede realizar para confirmar el fenotipo EMT. Por ejemplo, N-cadherina y α-actina de músculo liso se pueden teñir para un 29,31,32 fenotipo mesenquimal. Reorganización de la estructura del citoesqueleto se puede controlar mediante tinción de las células con faloidina para F-actina 11. Además, los ensayos para la motilidad celular y la resistencia a la muerte celular se pueden realizar para examinar las propiedades de EMT 11. 3. Detección de Empalme isoformas utilizando ensayos de qRT-PCR <ol><li> Diseño Primer NOTA: Primer pares deben ser cuidadosamente diseñados para amplificar distintas isoformas de empalme. Omisión de exón, el evento splicing alternativo más comúnmente observado, se muestra aquí como un ejemplo para ilustrar la estrategia de diseño del cebador. Figura 1A representa una de cuatro exón pre-ARNm, con el tercer exón como un exón variable. La inclusión del exón 3 resultados enla producción de una isoforma de empalme más largo, mientras que la omisión del exón 3 genera una isoforma de empalme más corta. Los cebadores deben ser diseñados para distinguir la isoforma exón-3-y-incluido el exón 3-omitido isoforma, y ​​para detectar la transcripción de ARNm total de este. <ol><li> Para la detección de exón inclusión, diseñar cuidadosamente un cebador directo dentro de la variable de exón 3, y un cebador inverso en el interior del exón constitutiva cerca 4, lo que permite la amplificación específica de la isoforma exón-incluido variable. Asegúrese de no diseñar adelante y atrás primers dentro del mismo exón variable con el fin de evitar los productos de PCR amplificados a partir de ADN genómico o pre-mRNA. </li><li> Para amplificar específicamente eventos omisión de exón, diseño uno de los cebadores que abarcan la unión del exón constitutiva 2 y el exón 4 que de otro modo se destruye cuando el exón variable de 3 se incluye. Diseñar el otro cebador dentro del exón constitutiva 4. Como tal, los productos PCR se generan sólo cuando el exón variable de 3 es en excluired y la constitutiva posteriormente se unen exón 2 y el exón 4 juntos. </li><li> Para detectar los transcritos totales del gen, el diseño de los cebadores dentro de los dos exones constitutivos 1 y 2 Por lo tanto, los productos de PCR se amplificaron a partir de todas las isoformas. </li><li> Asegúrese de que la temperatura de fusión (Tm) para todos los cebadores es de aproximadamente 55 ° C, la longitud de cada cebador es de aproximadamente 18-22 nucleótidos, y el tamaño de los productos de PCR es de entre 80 y 150 pares de bases. </li></ol></li><li> La extracción de RNA: Extracto de ARN utilizando un kit de aislamiento total de ARN (véase la Tabla de Materiales). <ol><li> Recoger las células en 350 l de tampón de lisis de ARN. </li><li> Añadir 350 l de etanol al 70% al lisado y mezclar bien. </li><li> Transferir la muestra a la columna de purificación de ARN. </li><li> Centrifugar a 10.000 xg durante 1 min y descarte de flujo a través. </li><li> Lavar la columna una vez con 500 l de tampón de lavado ARN I y dos veces con tampón de lavado II de ARN. </li><li> Retirar el tampón de lavado residual de ARNII de la columna por centrifugación a velocidad máxima durante 2 min. </li><li> Transferir la columna en un nuevo tubo de 1,5 ml, añadir 50 l de agua libre de nucleasa en el centro de la matriz de la columna y eluir el ARN por centrifugación a la velocidad máxima. </li></ol></li><li> Síntesis de la primera cadena de ADNc <ol><li> Determinar la concentración y la calidad del ARN mediante medición de la absorción UV. NOTA: Good ARN de calidad debe tener una relación de absorbancia / 280 nm 260 nm de aproximadamente 2,0. </li><li> Configuración de la transcriptasa inversa (RT) reacciones que contienen 250 ng de ARN total 0,05 g cebadores hexámeros al azar, 50 pmol de MgCl 2, 10 pmol dNTP, 2 l 5 x tampón de goscript, y 1 l de la enzima RT en un volumen final de 20 l. </li><li> Realizar RT reacciones a 42 º C durante 1 hora, seguido de incubación a 70 ° C durante 5 minutos para inactivar la enzima RT. </li></ol></li><li> PCR en tiempo real <ol><li> Establezca 20 l reacciones que consisten en 10 l SYBR verde Master Mix,0,1-1,0 l molde de ADNc, y 5 pmol cebadores directo e inverso. </li><li> Ejecute PCR en tiempo real con 40 ciclos de los siguientes pasos: 95 ° C desnaturalización durante 10 segundos, 58 ° C recocido durante 10 segundos, y 60 ° C de extensión durante 30 segundos. Lleve a cabo reacciones de PCR por triplicado. </li><li> Compruebe curvas de disociación y asegúrese de que un solo pico se observa para cada grupo de cebadores. </li><li> Obtener los valores de ciclo de cuantificación (Cq) mediante el cálculo de los segundos valores derivados de curvas de amplificación. </li><li> Calcular la cantidad relativa (RQ) de ARNm diana en las muestras inducidas en comparación con la muestra no inducida a través del &quot;delta delta Cq (ΔΔCq) &#39;método como se muestra por la siguiente fórmula. Utilice los genes de limpieza, tales como GAPDH o PDD, como referencia. </li></ol></li></ol><img alt="Ecuación 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Para calcular con mayor precisión los valores relativos de las isoformas de empalme, tl uso de múltiples genes de referencia debe ser considerada. Los resultados pueden ser analizados usando un método de cuantificación valor absoluto modificado descrito por Pfaffl et al 33,34. 4. Examen de Empalme Isoformas a nivel de proteínas <ol><li> Recoger las células en 100 tampón RIPA helada l. </li><li> Conjunto lisados ​​en hielo durante aproximadamente 10 min. </li><li> Centrifugar a 14.000 xg durante 10 min a 4 ° C, y recoger el sobrenadante. </li><li> Medir la concentración de proteína mediante ensayos de Bradford. </li><li> Diluir las muestras de proteína en tampón de carga SDS y cargar 20-40 g de proteínas en 10% geles de SDS-PAGE. </li><li> Ejecute geles SDS-PAGE a 20 mA durante 1,5 horas. </li><li> Proteínas de transferencia a membranas de PVDF en un sistema de transferencia húmeda a 4 ° C durante 3 horas a 85 V. Ajuste el tiempo de transferencia de acuerdo con el peso molecular de las proteínas diana. </li><li> Bloquear la membrana en la leche desnatada al 5% durante 30 min a 1 hora a TA. </li><li> Incubar wi membranath un anticuerpo primario a 4 ° CO / N. El rango de dilución de anticuerpo primario es normalmente entre 1: 200 y 1: 5000, determinar la dilución óptima para cada anticuerpo experimentalmente. La dilución de los anticuerpos utilizados en este protocolo se puede encontrar en la &quot;Tabla de reactivos y equipos específicos&quot;. <ol><li> Para detectar isoformas de empalme, utilizar anticuerpos específicos que reconocen una isoforma particular. Alternativamente, utilizar un anticuerpo que reconoce el epítopo en la región de codificación de exón constitutiva y detectar isoformas de empalme de forma simultánea sobre la base de sus diferentes tamaños de proteína. </li><li> Al mismo tiempo, controlar EMT por inmunotransferencia para los marcadores de EMT. Utilice E-cadherina, γ-catenina, y ocludina como marcadores epiteliales, y la fibronectina, N-cadherina y vimentina como marcadores mesenquimales. </li></ol></li><li> De membrana de lavado tres veces con TBS-T (Tris 50 mM, NaCl 150 mM, 0,05% de Tween 20, pH 7,4) en un intervalo de 5 min. </li><li> Incubar la membrana con una hormiga secundario conjugado con HRPibody en una dilución de 1: 10.000 durante 1 hora a RT. </li><li> Membrana Lavar tres veces con TBS-T en un intervalo de 5 min. </li><li> Visualizar proteína con un sistema de detección de quimioluminiscencia y exponer la membrana a una película de autorradiografía. </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.

El procedimiento descrito aquí permite la detección de corte y empalme alternativo en un modelo inducible EMT. Como tales, las alteraciones dinámicas de empalme isoforma expresión pueden ser capturados durante todo el curso de tiempo de EMT. Este método tiene ventajas sobre el uso de diferentes epithelial- o líneas de células mesenquimales-que representa para la comparación de splicing alternativo debido a antecedentes genéticos distintos de líneas celulares no relacionados, podrían influir indebidamente en e...

La transición epitelial-mesenquimal (EMT) es un programa de desarrollo que impulsa la morfogénesis de órganos y la remodelación de tejidos durante la embriogénesis. Cuando se activa de forma anormal, EMT promueve la metástasis tumoral y la fibrosis de órganos 1,2. Estudios convincentes han descrito la importancia de la regulación transcripcional durante el proceso de la EMT, que se define por varios factores de transcripción, como Twist, Caracol, y ZEB, que reprimen la expresión de la proteína de unión adherens E-cadherina, lo que resulta en la pérdida de un cobble- piedra, como la morfología del epitelio y la ganancia de un fenotipo mesenquimal fusiforme 3-8. Estudios recientes a través del análisis de todo el genoma de ARN revelaron que existe un grupo de genes cuyos patrones de empalme están asociados ya sea con fenotipos epiteliales o mesenquimales 9,10. El trabajo de nuestro laboratorio funcionalmente conectado splicing alternativo y EMT. Mediante el estudio de la superficie celular molécula de adhesión CD44, hemos demostrado que CD44 alternativa empalme está estrechamente regulada durante EMT, y lo más importante, que la isoforma de empalme CD44 conmutación causalmente contribuye a EMT 11. Splicing alternativo representa un modelo generalizado y conservado de la regulación de genes, ya que hasta el 95% de los genes de varios exones humanos están empalmados alternativamente 12-14. Mediante la generación de múltiples productos de proteína a partir de un único gen, corte y empalme alternativo constituye un mecanismo esencial para la diversidad de proteínas, la adición de otra capa de complejidad al genoma humano. Como tal, la desregulación de splicing alternativo podría dar lugar a profundos efectos biológicos que causan enfermedades humanas. De hecho, el splicing alternativo aberrante en enfermedades se ha documentado desde hace más de una década 15-25, incluyendo los recientes hallazgos de que las mutaciones en los genes que codifican la maquinaria spliceosome se encuentran comúnmente en los síndromes mielodisplásicos 26-28. Por lo tanto, el desarrollo de métodos fiables para la detección de i empalmados alternativamentesoforms es de gran importancia en el estudio de diversos procesos biológicos incluyendo la EMT. Aquí proporcionamos un protocolo para detectar cambios en el splicing alternativo utilizando un modelo EMT inducible. Los métodos para diseñar cebadores de PCR y la detección de isoformas de empalme son adecuados no sólo para el estudio de corte y empalme alternativo durante la EMT, sino también para el estudio de splicing alternativo en otros procesos biológicos. La investigación de corte y empalme alternativo durante EMT es imperativo con el fin de comprender mejor los mecanismos de la EMT y la metástasis del tumor, facilitando así el desarrollo de estrategias eficaces para tratar la metástasis del cáncer.

<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.

Los procedimientos descritos anteriormente proporcionan un método robusto para detectar splicing alternativo durante la EMT. Los resultados representativos de empalme isoforma CD44 conmutación durante EMT inducida Twist-A continuación se dan como ejemplo. EMT inducida Twist-en las células HMLE / Twist-ER se caracteriza por una transición de un adoquín como fenotipo epitelial a un fenotipo fibroblástico alargada (Figura 2A), la ausencia de marcadores epiteliales E-cadh...

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.

Detección de splicing alternativo Durante epitelial-mesenquimal de transición

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

12.9K vistas.  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.

Luz / oscuridad de transición de prueba para ratones

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, ...

Investigación

Biología

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.

La detección de ubiquitinación de proteínas

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

Detection of Functional Matrix Metalloproteinases by Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

La detección de metaloproteinasas de la matriz funcional por Zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

La detección de modificaciones de las histonas en hojas de las plantas

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

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-parámetro de medición de la apertura del poro de transición de permeabilidad en el corazón del ratón mitocondrias aisladas

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.

El análisis genético rápido de epitelio-mesenquimal de señalización durante la regeneración del cabello

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.

Inducción y Análisis de epitelial a mesenquimal de transición

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.

Culturas alternativas para Pluripotentes Stem Cell Production, mantenimiento y análisis genético

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.

Métodos para descubrir uso promotor alternativo y la regulación transcripcional de murino 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 enfoque alternativo para la preparación de muestras con bajo número de celular para el análisis TEM

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