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 Cultura de Células da EMT Indução Nota: EMT pode ser induzida por meio de tratamento de TGF-p ou expressão ectópica de factores de transcrição de torção, Snail, ou ZEB1 / 2 em células epiteliais. Descrito neste protocolo é um sistema EMT induzida via expressão da proteína de fusão Twist-ER nas células mamárias humanas imortalizadas epiteliais (HMLE / Twist-ER, um presente do Dr. J Yang, UCSD) 9,11,29. Após o 4-hidroxitamoxifeno (TAM) de tratamento, a proteína de fusão Twist-ER transloca-se para o núcleo para dirigir a transcrição e resulta numa transição EMT completo em 12-14 dias 9,11,29. Sistemas induzíveis EMT adicionais, como HMLE / Caracol-ER, HMLE / TGF e MCF10A / TGF, pode ser encontrada em nossas publicações anteriores 11,30. <ol><li> Manter células HMLE / tvist ER mamária em meio de crescimento de células epiteliais sem soro (MEGM) em uma incubadora a 37 ° C com 5% de CO 2. Passagem das células a cada 2-3 dias, quando chegar a 80% de confluência. Incubar as células com tripsina a 0,15% a 37 ° C durante 5-10 minutos, e, em seguida, inactivar a tripsina com 5% de soro de vitelo / DMEM. Gire as células e ressuspender-los em MEGM. Células da placa em uma nova placa de cultura de tecidos a uma razão de 1 a 4. </li><li> Dissolve-se em etanol a TAM para fazer uma solução 200 uM. Proteger da luz e da TAM armazenar a -20 ° C. Antes da utilização, adicionar recém TAM para os meios de MEGM a uma diluição de 1: 10000 para fazer uma concentração final de 20 nM a TAM. </li><li> Para a indução da EMT, placa células 1,6 x 10 6 HMLE / Torção-ER em um 10 centímetros de cultura de tecidos prato em duplicado. Células de cultura com 20 nM mídia TAM contendo MEGM. </li><li> Placa células 1,6 x 10 6 HMLE / Torção-ER em uma placa de 10 cm em duplicado e tratar as células com etanol 0,01% como controle tratados com não-TAM. </li><li> Dois dias após a incubação, para tirar fotos sob um microscópio de luz em aumento de 10x para gravar alterações morfológicas de células. </li><li> Aspirar o meio de um prato de controlo ou tratados com a TAMcélulas. Lavar as células com PBS frio, e as células da colheita pela demolição metade da placa em tampão de lise de ARN para o isolamento de ARN e a outra metade em tampão RIPA para análise de proteínas. </li><li> Células passagem do controle ou pratos tratados com TAM por re-chapeamento de 1,6 x 10 6 células em um prato de 10 cm em duplicado. Continuamente tratar as células com 20 nM de TAM ou veículo. </li><li> Repita os passos 5-7 em dias alternados até ao dia 14, altura em que as células tratadas Twist-ER-TAM mostram uma morfologia mesenquimal em forma de fuso, ao passo que as células de controlo tratadas com veículo a manutenção da morfologia epitelial de tipo paralelepípedo. </li></ol> 2 Caracterização de EMT Nota: A conclusão da EMT é indicado por: (1) Celulares alterações morfológicas de uma morfologia epitelial calçada semelhante a uma aparência de fibroblastos-like fusiforme; (2) Perda de localização de E-caderina nas junções célula-célula; e (3) a expressão dos marcadores de comutação EMT, definida pela perdade marcadores epiteliais e ganho de marcadores mesenquimais. Celulares alterações morfológicas são captadas por microscópio de luz em aumento de 10x durante o curso do tempo de indução EMT, descrito na Seção 1 detecção de Imuno-fluorescência de E-caderina em junções celulares é descrita nesta seção. A expressão dos marcadores de EMT é detectada por imunotransferência. Marcadores epiteliais comuns são a E-caderina, γ-catenina, e ocludina e marcadores mesenquimais incluem fibronectina, N-caderina e vimentina. Procedimentos gerais de immunoblotting estão descritos na Seção 4. <ol><li> No dia 12 do tratamento TAM, semente de 1 x 10 5 células em um de 12 milímetros de vidro lamela circular colocado num poço de uma placa de 24 poços. </li><li> 48 horas depois que as células de semeadura, médias Aspirar e lavar com pré-aquecido PBS. </li><li> Fix células com pré-aquecido a 4% de paraformaldeído durante 15 min. </li><li> Lave as células três vezes com PBS. </li><li> Incubar as células com 0,2% de Triton X-100 em PBS por 15min à temperatura ambiente. </li><li> Lave as células três vezes com PBS. </li><li> Incubar as células em 5% de BSA / PBS, durante 1 hora à temperatura ambiente para bloquear a ligação não específica. </li><li> Incubar as células com o anticorpo de E-caderina primário numa diluição 1:50 em BSA a 1% / PBS em uma câmara humidificada S / N a 4 ° C. NOTA: O intervalo para a diluição de anticorpo primário é normalmente entre 1:50 e 1: 200, e a diluição óptima para cada anticorpo deve ser determinada experimentalmente. </li><li> Decantar o anticorpo primário e lava-se as células três vezes com PBS. </li><li> Incubar as células com o anticorpo secundário marcado com fluorescência em uma diluição 1: 500, durante 1 h à TA. A partir deste passo, a proteger as células da luz. </li><li> Decantar o anticorpo secundário e lava-se três vezes com PBS. </li><li> Células mancha com 0,1-1 g / ml DAPI ou Hoechst para 10 min. </li><li> Lavar as células com PBS três vezes. </li><li> Monte lamelas com uma gota de meio de montagem, e lamelas de vedação com unha polonês. </li><li> Observar células e tomarimagens em um microscópio fluorescente 63X. </li></ol> A coloração com anticorpos adicionais podem ser realizados para confirmar o fenótipo de EMT. Por exemplo, N-caderina e actina de músculo liso-α pode ser corado por um 29,31,32 fenótipo mesenquimal. Reorganização de estrutura do citoesqueleto podem ser monitorizadas por coloração das células com faloidina para F-actina 11. Além disso, ensaios para a motilidade das células e resistência à morte celular podem ser realizados para examinar as propriedades dos EMT 11. 3 Detecção de Splice isoformas utilizando qRT-PCR Ensaios <ol><li> Projeto Primer NOTA: Os pares de primers devem ser cuidadosamente projetado para amplificar isoformas de splicing distintas. Exon pular, o acontecimento de processamento alternativo mais frequentemente observados, é mostrado como um exemplo para ilustrar a estratégia de desenho de primers. Figura 1A ilustra um exão de quatro pré-ARNm, com o terceiro exão de um exão variável. Inclusão de exão 3 resultaa produção de uma isoforma de emenda mais tempo, enquanto que o salto do exon 3 gera uma isoforma mais curta de splicing. Os iniciadores devem ser concebidos para distinguir a isoforma incluído exão-3-e isoforma do exão-3-ignorado, e para detectar o transcrito total deste ARNm. <ol><li> Para a detecção de inclusão exão, desenhar cuidadosamente um iniciador para a frente no interior do exão 3 variável, e uma sequência iniciadora reversa no interior do exão 4 constitutiva vizinhas, permitindo a amplificação específica da isoforma variável incluído-exão. Certifique-se não tanto à frente e conceber os iniciadores no seio da mesma exão variável inversa, a fim de evitar produtos de PCR amplificados a partir de ADN genómico ou de pré-ARNm. </li><li> Para amplificar especificamente os eventos ignorando exão, design um dos iniciadores, abrangendo a junção do exão 2 e constitutiva exão 4, que de outra forma seriam destruídos quando exão variável 3 está incluído. Conceber o outro iniciador no interior do exão 4 constitutiva Como tal, os produtos de PCR são gerados apenas quando o exão 3 é variável, excluied eo constitutivo exon 2 e exon 4 são posteriormente unidas. </li><li> Para detectar as transcrições totais do gene, desenhar os iniciadores de dentro de dois exons constitutivos 1 e 2 Assim, os produtos de PCR são amplificados a partir de todas as isoformas. </li><li> Certifique-se de que a temperatura de fusão (Tm) para todos os iniciadores é de aproximadamente 55 ° C, a duração de cada um dos iniciadores é aproximadamente 18-22 nucleótidos, e o tamanho dos produtos de PCR é entre 80 e 150 pares de bases. </li></ol></li><li> Extração de RNA: Extrato RNA usando um kit de isolamento total RNA (ver Tabela de Materiais). <ol><li> Recolher as células em 350 ul de tampão de lise de ARN. </li><li> Adicionar 350 mL de etanol a 70% ao ligado e misture bem. </li><li> Transfira a amostra para a coluna de purificação de ARN. </li><li> Centrifuga-se a 10000 xg durante 1 min e descarte de fluxo. </li><li> Lava-se a coluna uma vez com 500 ul de tampão de lavagem de RNA I e duas vezes com tampão de lavagem de ARN II. </li><li> Remover o tampão de lavagem residual ARNII a partir da coluna através de centrifugação à velocidade máxima durante 2 min. </li><li> Transferir a coluna para um novo tubo de 1,5 ml, adicionar 50 mL de água isenta de nuclease, no centro da matriz da coluna e eluir o ARN por centrifugação à velocidade máxima. </li></ol></li><li> Síntese da primeira cadeia de cDNA <ol><li> Determinar a concentração e a qualidade do RNA por medição da absorção de UV. NOTA: qualidade RNA bom deve ter uma relação absorbância / 280 nm 260 nm de aproximadamente 2,0. </li><li> Defina-se a transcriptase reversa (RT), de reacções que contêm 250 ng de ARN total, a 0,05 ug iniciadores de hexâmeros ao acaso, 50 pmol de MgCl2, 10 pmol de dNTPs, 2 ul de tampão 5 × GoScript, e 1 ul da enzima RT, num volume final de 20 ul. </li><li> Realizar as reacções de RT a 42 ° C durante 1 hora, seguido de incubação a 70 ° C durante 5 min, para inactivar a enzima RT. </li></ol></li><li> PCR em tempo real <ol><li> Configure 20 reacções ul que consistem em 10 mL SYBR master mix verde,0,1-1,0 modelo de cDNA ul e 5 pmol de iniciadores directos e inversos. </li><li> Executar PCR em tempo real com 40 ciclos dos seguintes passos: 95 ° C desnaturação durante 10 seg, 58 ° C, emparelhamento durante 10 segundos, e extensão a 60 ° C durante 30 seg. Realizar reações de PCR em triplicado. </li><li> Confira curvas de dissociação e certifique-se que um único pico é observado para cada conjunto de primers. </li><li> Obtenção dos valores de ciclo de quantificação (Cq), calculando os segundos valores derivados das curvas de amplificação. </li><li> Calcula-se a quantidade relativa (RQ) de mRNAs alvo em amostras induzidas, em comparação com a amostra não induzido utilizando o &quot;delta delta Cq (ΔΔCq) &#39;método, como mostrado pela seguinte fórmula. Use genes de manutenção, tais como GAPDH ou TBP, como referência. </li></ol></li></ol><img alt="Equação 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> Para calcular com mais precisão os valores relativos das isoformas de splicing, tele utilizar de múltiplos genes de referência deverá ser considerada. Os resultados podem ser analisados ​​utilizando um método de quantificação de valor absoluto modificado descrito por Pfaffl et al 33,34. 4. Exame da Splice isoformas no nível de proteína <ol><li> Recolher as células em 100 de tampão RIPA arrefecido em gelo ul. </li><li> Definir lisados ​​em gelo durante cerca de 10 min. </li><li> Centrifuga-se a 14000 xg durante 10 min a 4 ° C, e recolher o sobrenadante. </li><li> Medir a concentração de proteína por meio de ensaios de Bradford. </li><li> Diluir as amostras de proteína em tampão de carga de SDS e carregar 20-40 ug de proteínas em géis de 10% SDS-PAGE. </li><li> Execute géis SDS-PAGE a 20 mA durante 1,5 horas. </li><li> Proteínas de transferência para membranas de PVDF em um sistema de transferência húmida a 4 ° C durante 3 horas a 85 V. Ajustar o tempo de transferência de acordo com o peso molecular de proteínas alvo. </li><li> Bloco de membrana em 5% de leite desnatado durante 30 minutos a 1 hora a RT. </li><li> Incubar wi membranapo um anticorpo primário a 4 ° CO / N. O intervalo para a diluição de anticorpo primário é normalmente entre 1: 200 e 1: 5000, determinar a diluição óptima para cada anticorpo experimentalmente. A diluição dos anticorpos usados ​​no presente protocolo podem ser encontrados em &quot;Tabela de reagentes e equipamentos específicos&quot;. <ol><li> Para detectar as isoformas de splicing, utilizar anticorpos específicos que reconhecem uma isoforma particular. Em alternativa, utilizar um anticorpo que reconhece o epitopo na região de codificação de exão constitutiva e detectar as isoformas de splicing, simultaneamente, com base nos seus tamanhos diferentes de proteína. </li><li> Ao mesmo tempo, controlar EMT por imunotransferência por marcadores EMT. Use E-caderina, γ-catenina, e ocludina como marcadores epiteliais, e fibronectina, N-caderina e vimentina como marcadores mesenquimais. </li></ol></li><li> Membrana de lavagem três vezes com TBS-T (50 mM Tris, 150 mM de NaCl, 0,05% de Tween 20, pH 7,4) em um intervalo de 5 minutos. </li><li> Incubar membrana com uma formiga secundário conjugado com HRPibody em uma diluição de 1: 10000 a 1 hora à temperatura ambiente. </li><li> Membrana de lavagem três vezes com TBS-T com um intervalo de 5 minutos. </li><li> Visualizar proteína com um sistema de detecção de quimioluminescência e expor a membrana a uma película de auto-radiografia. </li></ol>

<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.

O processo aqui descrito permite a detecção de splicing alternativo de um modelo de EMT indutível. Como tal, as alterações dinâmicas de emenda de expressão da isoforma pode ser captado em todo o curso de tempo de EMT. Este método tem vantagens sobre o uso de diferentes epithelial- ou linhas de células-representando mesenquimais para a comparação de splicing alternativo, porque origens genéticas distintas de linhas celulares relacionadas un poderia influenciar indevidamente splicing alternativo. No entanto, a...

A transição epitelial-mesenquimal (EMT) é um programa de desenvolvimento que impulsiona a morfogênese de órgãos e remodelação do tecido durante a embriogênese. Quando activados anormalmente, EMT promove a metástase do tumor e órgão fibrose 1,2. Obrigando estudos descreveram a importância da regulação da transcrição durante o processo de EMT, definida por vários factores de transcrição, tais como torção, Snail, e ZEB, que reprimem a expressão da proteína de junção aderente E-caderina, que resultam na perda de um cobble- pedra, como morfologia epitelial e ganho de um fenótipo mesenquimal fusiforme 3-8. Estudos recentes por meio de análise de todo o genoma de ARN revelou que existe um grupo de genes cujos padrões de splicing estão associadas quer epiteliais ou mesenquimais fenótipos 9,10. Trabalho de nosso laboratório de funcionalmente ligados splicing alternativo e EMT. Ao estudar a molécula de adesão de superfície celular CD44, foi demonstrado que CD44 alternative splicing é rigidamente controlado durante a EMT, e mais importante, que isoforma CD44 emenda comutação causalmente contribui para EMT 11. O splicing alternativo representa um modelo disseminado e conservada da regulação de genes, tal como até 95% de genes de multi-exões humanos são de splicing alternativo 12-14. Através da produção de vários produtos de proteínas a partir de um único gene, o splicing alternativo constitui um mecanismo essencial para a diversidade de proteínas, a adição de uma outra camada de complexidade para o genoma humano. Como tal, a desregulação de splicing alternativo pode potencialmente levar a profundos efeitos biológicos que causam doenças humanas. Na verdade, o splicing alternativo aberrante em doenças tem sido documentada por mais de uma década de 15-25, incluindo as recentes descobertas de que as mutações em genes que codificam a maquinaria spliceosome são comumente encontrados em síndromes mielodisplásicas 26-28. Portanto, o desenvolvimento de métodos confiáveis ​​para a detecção de i de splicing alternativosoforms é de grande importância no estudo de processos biológicos diversos, incluindo EMT. Aqui nós fornecemos um protocolo para detectar mudanças no splicing alternativo utilizando um modelo EMT induzida. Os métodos para a concepção de iniciadores de PCR e detectar as isoformas de splicing são adequadas não apenas para o estudo de splicing alternativo durante EMT, mas também para o estudo de splicing alternativo em outros processos biológicos. Investigando splicing alternativo durante EMT é imprescindível, a fim de compreender melhor os mecanismos da EMT ea metástase do tumor, facilitando, assim, o desenvolvimento de estratégias eficazes para tratar a metástase do câncer.

<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.

Os procedimentos descritos acima fornecem um método robusto para detectar splicing alternativo durante EMT. Os resultados representativos de CD44 splice isoforma de comutação durante EMT induzida por torção são dadas abaixo como um exemplo. EMT induzida por torção em células HMLE / twist-ER foi caracterizado por uma transição de uma calçada de pedra como fenótipo epitelial para um fenótipo fibroblástica alongada (Figura 2A), a ausência de marcadores epiteliai...

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.

Detecção de splicing alternativo Durante epitelial-mesenquimal Transição

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 visualizações.  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.

Vídeo: 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.

Claro / escuro Teste de transição para Ratos

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, ...

Biologia

Detection of Protein Ubiquitination

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

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

Detecção de Ubiquitinação Protein

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.

Detecção de Metaloproteinases da Matriz Funcional por zimografia

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.

Detecção de histona Modificações nas folhas das 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.

Parâmetro Multi-Medição da abertura dos poros transição de permeabilidade em mitocôndrias isoladas de coração Rato

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.

Análise Genética rápida de epitelial-mesenquimal sinalização durante a regeneração do cabelo

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.

Indução e Análise de epitelial para mesenquimal Transição

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 estaminais humanas produção de células, Manutenção e Análise Genética

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 descobrir Uso Promotor Alternativa e regulação da transcrição 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.

Uma abordagem alternativa para preparação de amostras com baixo número de celular de Análise TEM

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