Name: detection of alternative splicing during epithelial-mesenchymal transition
Uploaded: 2014-10-09T14:00:00
Description: Watch this Scientific Journal Video about Detection of Alternative Splicing During Epithelial-Mesenchymal Transition at JoVE.com

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. Cell Culture of EMT Induction
Note: EMT can be induced by treatment of TGF&#946; or ectopic expression of transcription factors Twist, Snail, or Zeb1/2 in epithelial cells. Described in this protocol is an inducible EMT system via expression of the Twist-ER fusion protein in immortalized human mammary epithelial cells (HMLE/Twist-ER, a gift of Dr. J Yang, UCSD)9,11,29. Upon 4-hydroxytamoxifen (TAM) treatment, the fusion protein Twist-ER translocates to the nucleus to drive transcri...

<ol>
	<li>Thiery, J. P., Sleeman, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complex+networks+orchestrate+epithelial-mesenchymal+transitions.">Complex networks orchestrate epithelial-mesenchymal transitions.</a> Nat Rev Mol Cell Biol. 7, 131-142 (2006).</li><li>Yang, J., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transition+at+the+crossroads+of+development+and+tumor+metastasis.">Epithelial-mesenchymal transition at the crossroads of development and tumor metastasis.</a> Dev Cell. 14, 818-829 (2008).</li><li>Yang, J., 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+master+regulator+of+morphogenesis,+plays+an+essential+role+in+tumor+metastasis.">a master regulator of morphogenesis, plays an essential role in tumor metastasis.</a> Cell. 117, 927-939 (2004).</li><li>Batlle, 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=The+transcription+factor+snail+is+a+repressor+of+E-cadherin+gene+expression+in+epithelial+tumour+cells.">The transcription factor snail is a repressor of E-cadherin gene expression in epithelial tumour cells.</a> Nat Cell Biol. 2, 84-89 (2000).</li><li>Cano, 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+transcription+factor+snail+controls+epithelial-mesenchymal+transitions+by+repressing+E-cadherin+expression.">The transcription factor snail controls epithelial-mesenchymal transitions by repressing E-cadherin expression.</a> Nat Cell Biol. 2, 76-83 (2000).</li><li>Comijn, J., 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+two-handed+E+box+binding+zinc+finger+protein+SIP1+downregulates+E-cadherin+and+induces+invasion.">The two-handed E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion.</a> Mol Cell. 7, 1267-1278 (2001).</li><li>Bolos, V., 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+transcription+factor+Slug+represses+E-cadherin+expression+and+induces+epithelial+to+mesenchymal+transitions:+a+comparison+with+Snail+and+E47+repressors.">The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors.</a> J Cell Sci. 116, 499-511 (2003).</li><li>Eger, 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=DeltaEF1+is+a+transcriptional+repressor+of+E-cadherin+and+regulates+epithelial+plasticity+in+breast+cancer+cells.">DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells.</a> Oncogene. 24, 2375-2385 (2005).</li><li>Shapiro, I. 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=An+EMT-driven+alternative+splicing+program+occurs+in+human+breast+cancer+and+modulates+cellular+phenotype.">An EMT-driven alternative splicing program occurs in human breast cancer and modulates cellular phenotype.</a> PLoS Genet. 7, e1002218 (2011).</li><li>Warzecha, C. C., 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=An+ESRP-regulated+splicing+programme+is+abrogated+during+the+epithelial-mesenchymal+transition.">An ESRP-regulated splicing programme is abrogated during the epithelial-mesenchymal transition.</a> EMBO J. 29, 3286-3300 (2010).</li><li>Brown, R. 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=CD44+splice+isoform+switching+in+human+and+mouse+epithelium+is+essential+for+epithelial-mesenchymal+transition+and+breast+cancer+progression.">CD44 splice isoform switching in human and mouse epithelium is essential for epithelial-mesenchymal transition and breast cancer progression.</a> J Clin Invest. 121, 1064-1074 (2011).</li><li>Wang, E. T., 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+isoform+regulation+in+human+tissue+transcriptomes.">Alternative isoform regulation in human tissue transcriptomes.</a> Nature. 456, 470-476 (2008).</li><li>Pan, Q., Shai, O., Lee, L. J., Frey, B. J., Blencowe, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+surveying+of+alternative+splicing+complexity+in+the+human+transcriptome+by+high-throughput+sequencing.">Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.</a> Nat Genet. 40, 1413-1415 (2008).</li><li>Liu, S., 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=Alternative+RNA+splicing+and+cancer.">Alternative RNA splicing and cancer.</a> Wiley Interdiscip Rev RNA. 4, 547-566 (2013).</li><li>Shtivelman, E., Lifshitz, B., Gale, R. P., Roe, B. A., Canaani, E. <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+RNAs+transcribed+from+the+human+abl+gene+and+from+the+bcr-abl+fused+gene.">Alternative splicing of RNAs transcribed from the human abl gene and from the bcr-abl fused gene.</a> Cell. 47, 277-284 (1986).</li><li>Krangel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+of+HLA-A+and+-B+antigens+via+an+alternative+RNA+splicing+pathway.">Secretion of HLA-A and -B antigens via an alternative RNA splicing pathway.</a> J Exp Med. 163, 1173-1190 (1986).</li><li>Brickell, P. M., Latchman, D. S., Murphy, D., Willison, K., Rigby, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+a+Qa/Tla+class+I+major+histocompatibility+antigen+gene+is+a+general+feature+of+oncogenesis+in+the+mouse.">Activation of a Qa/Tla class I major histocompatibility antigen gene is a general feature of oncogenesis in the mouse.</a> Nature. 306, 756-760 (1983).</li><li>Barbaux, S., 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=Donor+splice-site+mutations+in+WT1+are+responsible+for+Frasier+syndrome.">Donor splice-site mutations in WT1 are responsible for Frasier syndrome.</a> Nat Genet. 17, 467-470 (1997).</li><li>Charlet, B. 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=Loss+of+the+muscle-specific+chloride+channel+in+type+1+myotonic+dystrophy+due+to+misregulated+alternative+splicing.">Loss of the muscle-specific chloride channel in type 1 myotonic dystrophy due to misregulated alternative splicing.</a> Mol Cell. 10, 45-53 (2002).</li><li>Hutton, 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=Association+of+missense+and+5'-splice-site+mutations+in+tau+with+the+inherited+dementia+FTDP-17.">Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17.</a> Nature. 393, 702-705 (1998).</li><li>Kohsaka, T., 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=Exon+9+mutations+in+the+WT1+gene,+without+influencing+KTS+splice+isoforms,+are+also+responsible+for+Frasier+syndrome.">Exon 9 mutations in the WT1 gene, without influencing KTS splice isoforms, are also responsible for Frasier syndrome.</a> Hum Mutat. 14, 466-470 (1999).</li><li>Philips, A. V., Timchenko, L. T., 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=Disruption+of+splicing+regulated+by+a+CUG-binding+protein+in+myotonic+dystrophy.">Disruption of splicing regulated by a CUG-binding protein in myotonic dystrophy.</a> Science. 280, 737-741 (1998).</li><li>Savkur, R. S., Philips, A. V., 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=Aberrant+regulation+of+insulin+receptor+alternative+splicing+is+associated+with+insulin+resistance+in+myotonic+dystrophy.">Aberrant regulation of insulin receptor alternative splicing is associated with insulin resistance in myotonic dystrophy.</a> Nat Genet. 29, 40-47 (2001).</li><li>Spillantini, M. G., 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=Mutation+in+the+tau+gene+in+familial+multiple+system+tauopathy+with+presenile+dementia.">Mutation in the tau gene in familial multiple system tauopathy with presenile dementia.</a> Proc Natl Acad Sci USA. 95, 7737-7741 (1998).</li><li>Wang, J., 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=Myotonic+dystrophy+evidence+for+a+possible+dominant-negative+RNA+mutation.">Myotonic dystrophy evidence for a possible dominant-negative RNA mutation.</a> Hum Mol Genet. 4, 599-606 (1995).</li><li>Graubert, T. 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 procedure described here enables the detection of alternative splicing in an inducible EMT model. As such, dynamic alterations of splice isoform expression can be captured throughout the time course of EMT. This method has advantages over the use of different epithelial- or mesenchymal-representing cell lines for comparison of alternative splicing because distinct genetic backgrounds from un-related cell lines could unduly influence alternative splicing. However, the successful induction of EMT must be carefully conf...

The epithelial-mesenchymal transition (EMT) is a developmental program that drives organ morphogenesis and tissue remodeling during embryogenesis. When abnormally activated, EMT promotes tumor metastasis and organ fibrosis1,2. Compelling studies have described the importance of transcriptional regulation during the process of EMT, defined by several transcription factors, such as Twist, Snail, and ZEB, which repress the expression of the adherens junction protein E-cadherin, resulting in loss of a cobble-stone like epithelial morphology and gain of a spindle-shaped mesenchymal phenotype3-8. Recent studies through genome-wide analysis of RNAs revealed that there exists a group of genes whose splicing patterns are associated with either epithelial or mesenchymal phenotypes9,10. Work from our lab functionally connected alternative splicing and EMT. By studying the cell surface adhesion molecule CD44, we demonstrated that CD44 alternative splicing is tightly regulated during EMT, and more importantly, that CD44 splice isoform switching causally contributes to EMT11.
Alternative splicing represents a widespread and conserved model of gene regulation, as up to 95% of human multi-exon genes are alternatively spliced12-14. By generating multiple protein products from a single gene, alternative splicing constitutes an essential mechanism for protein diversity, adding another layer of complexity to the human genome. As such, dysregulation of alternative splicing could potentially lead to profound biological effects causing human diseases. Indeed, aberrant alternative splicing in diseases has been documented for over a decade15-25, including recent findings that mutations in genes encoding the spliceosome machinery are commonly found in myelodysplastic syndromes26-28. Therefore, developing reliable methods for the detection of alternatively spliced isoforms is of great importance in the study of diverse biological processes including EMT.
Here we provide a protocol to detect changes in alternative splicing using an inducible EMT model. The methods for designing PCR primers and detecting splice isoforms are suitable not only for the study of alternative splicing during EMT, but also for the study of alternative splicing in other biological processes. Investigating alternative splicing during EMT is imperative in order to better understand the mechanisms of EMT and tumor metastasis, thus facilitating the development of effective strategies to treat cancer metastasis.

<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.

The procedures described above provide a robust method to detect alternative splicing during EMT. Representative results of CD44 splice isoform switching during Twist-induced EMT are given below as an example.
Twist-induced EMT in HMLE/Twist-ER cells was characterized by a transition from a cobble-stone like epithelial phenotype to an elongated fibroblastic phenotype (Figure 2A), the absence of epithelial markers E-cadherin, &#947;-catenin and occludin and the upregulation of ...

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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.

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

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