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 transcription and results in a full EMT transition in 12-14 days9,11,29. Additional EMT inducible systems, such as HMLE/Snail-ER, HMLE/TGF&#946;, and MCF10A/TGF&#946;, can be found in our previous publications11,30.
<ol>
	<li>Maintain HMLE/Twist-ER cells in serum-free mammary epithelial cell growth medium (MEGM) in a 37 &#176;C incubator with 5% CO2. Passage the cells every 2-3&#160;days when they reach 80% confluence. Incubate cells with 0.15% trypsin at 37 &#176;C for 5-10 min, and then inactivate trypsin with 5% calf serum/DMEM. Spin down cells and resuspend them in MEGM. Plate cells in a new tissue culture dish at a ratio of 1 to 4.</li>
	<li>Dissolve TAM in ethanol to make a 200 &#956;M solution. Protect TAM from light and store at -20 &#176;C. Before use, freshly add TAM into MEGM media at a 1:10,000 dilution to make a final concentration of 20 nM TAM.</li>
	<li>For induction of EMT, plate 1.6 x 106 HMLE/Twist-ER cells into a 10 cm tissue culture dish in duplicate. Culture cells with 20 nM TAM-containing MEGM media.</li>
	<li>Plate 1.6 x&#160;106 HMLE/Twist-ER cells into a 10 cm dish in duplicate and treat cells with 0.01% ethanol as a non-TAM-treated control.</li>
	<li>Two days after incubation, take pictures under a light microscope at 10X&#160;magnification to record morphological changes of cells.</li>
	<li>Aspirate the medium from one dish of control or TAM-treated cells. Wash cells with cold PBS, and harvest cells by scrapping half of the plate in RNA lysis buffer for RNA isolation and the other half in RIPA buffer for protein analysis.</li>
	<li>Passage cells from the control or TAM-treated dishes by re-plating 1.6 x&#160;106 cells in a 10 cm dish in duplicate. Continuously treat the cells with 20 nM TAM or vehicle.</li>
	<li>Repeat steps 5 to 7 once every other day until day 14, at which time, TAM-treated Twist-ER cells show a spindle-shaped mesenchymal morphology, whereas vehicle-treated control cells maintain the cobblestone-like epithelial morphology.</li>
</ol>
2. Characterization of EMT
Note: A completion of EMT is indicated by: (1) Cell morphological changes from a cobblestone-like epithelial morphology to a spindle-shaped fibroblastic-like appearance; (2) Loss of E-cadherin localization at cell-cell junctions; and (3) The switched expression of EMT markers, defined by the loss of epithelial markers and gain of mesenchymal markers.
Cell morphological changes are captured by light microscope at 10X&#160;magnification during the time course of EMT induction, described in Section 1. Immuno-fluorescence detection of E-cadherin at cell-junctions is described in this section. The expression of EMT markers is detected by immunoblotting. Common epithelial markers are E-cadherin, &#947;-catenin, and occludin, and mesenchymal markers include fibronectin, N-cadherin and vimentin. General procedures of immunoblotting are described in Section 4.
<ol>
	<li>At day 12 of TAM treatment, seed 1 x 105 cells on a 12 mm glass circular coverslip placed in a well of a 24-well plate.</li>
	<li>48 hr&#160;after seeding, aspirate medium and wash cells with pre-warmed PBS.</li>
	<li>Fix cells with pre-warmed 4% paraformaldehyde for 15 min.</li>
	<li>Wash cells three times with PBS.</li>
	<li>Incubate cells with 0.2% Triton X-100 in PBS for 15 min&#160;at RT.</li>
	<li>Wash cells three times&#160;with PBS.</li>
	<li>Incubate cells in 5% BSA/PBS for 1 hr at RT to block non-specific binding.</li>
	<li>Incubate cells with the primary antibody E-cadherin at a 1:50 dilution in 1% BSA/PBS in a humidified chamber O/N&#160;at 4 &#176;C. NOTE: The range for primary antibody dilution is normally between 1:50 and 1:200, and the optimal dilution for each antibody should be determined experimentally.</li>
	<li>Decant the primary antibody and wash cells three times&#160;with PBS.</li>
	<li>Incubate cells with fluorescent-labeled secondary antibody in a 1:500 dilution for 1 hr at RT. From this step on, protect cells from light.</li>
	<li>Decant the secondary antibody and wash three times&#160;with PBS.</li>
	<li>Stain cells with 0.1-1 g/ml DAPI or Hoechst for 10 min.</li>
	<li>Rinse cells with PBS three times.</li>
	<li>Mount coverslips with a drop of mounting medium, and seal coverslips with nail polish.</li>
	<li>Observe cells and take images under a 63X&#160;fluorescent microscope.</li>
</ol>
Staining with additional antibodies can be performed to confirm the EMT phenotype. For example, N-cadherin and &#945;-smooth muscle actin can be stained for a mesenchymal phenotype29,31,32. Reorganization of cytoskeletal structure can be monitored by staining cells with phalloidin for F-actin11. In addition, assays for cell motility and cell-death resistance can be performed to examine the properties of EMT11.
3. Detection of Splice Isoforms Using qRT-PCR Assays
<ol>
	<li>Primer Design 
	NOTE: Primer pairs should be carefully designed to amplify distinct splice isoforms. Exon skipping, the most commonly observed alternative splicing event, is shown here as an example to illustrate the strategy of primer design. Figure 1A depicts a four-exon pre-mRNA, with the third exon as a variable exon. Inclusion of exon 3 results in the production of a longer splice isoform, whereas skipping of exon 3 generates a shorter splice isoform. Primers need to be designed to distinguish the exon-3-included isoform and exon-3-skipped isoform, and to detect the total transcript of this mRNA.
	<ol>
		<li>For detection of exon inclusion, carefully design a forward primer inside the variable exon 3, and a reverse primer inside the nearby constitutive exon 4, allowing for the specific amplification of the variable exon-included isoform. Make sure not to design both forward and reverse primers within the same variable exon in order to avoid PCR products amplified from genomic DNA or pre-mRNA.</li>
		<li>To specifically amplify exon skipping events, design one of the primers spanning the junction of the constitutive exon 2 and exon 4 that would otherwise be destroyed when variable exon 3 is included. Design the other primer inside the constitutive exon 4. As such, PCR products are generated only when the variable exon 3 is excluded and the constitutive exon 2 and exon 4 are subsequently joined together.</li>
		<li>To detect the total transcripts of the gene, design the primers within two constitutive exons 1 and 2. Thus, PCR products are amplified from all isoforms.</li>
		<li>Make sure the melting temperature (Tm) for all primers is approximately 55 &#176;C, the length of each primer is approximately 18-22 nucleotides, and the size of PCR products is between 80 and 150 base pairs.</li>
	</ol>
	</li>
	<li>RNA extraction: Extract RNA using a total RNA isolation kit (see Table of Materials).
	<ol>
		<li>Collect cells in 350 &#956;l RNA lysis buffer.</li>
		<li>Add 350 &#956;l of 70% ethanol to the lysate and mix thoroughly.</li>
		<li>Transfer the sample to the RNA purification column.</li>
		<li>Centrifuge at 10,000 x&#160;g for 1 min&#160;and discard flow-through.</li>
		<li>Wash the column once with 500 &#956;l of RNA wash buffer I and twice with RNA wash buffer II.</li>
		<li>Remove residual RNA wash buffer II from the column by centrifugation at maximum speed for 2 min.</li>
		<li>Transfer the column into a new 1.5 ml tube, add 50 &#956;l nuclease-free water at the center of the column matrix and elute RNA by centrifugation at the maximum speed.</li>
	</ol>
	</li>
	<li>Synthesis of First-strand cDNA
	<ol>
		<li>Determine the concentration and quality of RNA by UV absorption measurement. NOTE: Good quality RNA should have a 260 nm / 280 nm absorbance ratio of approximately 2.0.</li>
		<li>Set up reverse transcriptase (RT) reactions that contain 250 ng total RNA, 0.05 &#956;g random hexamer primers, 50 pmol MgCl2, 10 pmol dNTP, 2 &#956;l 5&#215; GoScript buffer, and 1 &#956;l RT enzyme in a final volume of 20 &#956;l.</li>
		<li>Perform RT reactions at 42 &#176;C for 1 hr, followed by incubation at 70 &#176;C for 5 min&#160;to inactivate the RT enzyme.</li>
	</ol>
	</li>
	<li>Real-time PCR
	<ol>
		<li>Set up 20 &#956;l reactions that consist of 10 &#956;l SYBR green master mix, 0.1-1.0 &#956;l cDNA template, and 5 pmol forward and reverse primers.</li>
		<li>Run real-time PCR with 40 cycles of the following steps: 95 &#176;C denaturation for 10 sec, 58 &#176;C annealing for 10 sec, and 60 &#176;C extension for 30 sec. Perform PCR reactions in triplicate.</li>
		<li>Check dissociation curves and make sure that a single peak is observed for each primer set.</li>
		<li>Obtain the quantification cycle (Cq) values by calculating the second derivative values of amplification curves.</li>
		<li>Calculate the relative quantity (RQ) of target mRNAs in induced samples as compared to the uninduced sample using the &#8216;delta delta Cq (&#916;&#916;Cq)&#8217; method as shown by the following formula. Use housekeeping genes, such as GAPDH or TBP, as a reference.</li>
	</ol>
	</li>
</ol>
<img alt="Equation 1" fo:content-width="3in" src="/files/ftp_upload/51845/51845eq1.jpg" /> 
To more accurately calculate relative values of splice isoforms, the use of multiple reference genes should be considered. The results can be analyzed using a modified absolute value quantification method described by Pfaffl et al33,34.
4. Examination of Splice Isoforms at the Protein Level
<ol>
	<li>Collect cells in 100 &#956;l ice-cold RIPA buffer.</li>
	<li>Set lysates on ice for approximately 10 min.</li>
	<li>Centrifuge at 14,000 x g for 10 min&#160;at 4 &#176;C, and collect the supernatant.</li>
	<li>Measure protein concentration by Bradford assays.</li>
	<li>Dilute protein samples in SDS loading buffer and load 20-40 &#956;g of proteins into 10% SDS-PAGE gels.</li>
	<li>Run SDS-PAGE gels at 20 mA for 1.5 hr.</li>
	<li>Transfer proteins to PVDF membranes in a wet transfer system at 4 &#176;C for 3 hr&#160;at 85 V. Adjust the transfer time according to the molecular weight of target proteins.</li>
	<li>Block membrane in 5% non-fat milk for 30 min&#160;to 1 hr at RT.</li>
	<li>Incubate membrane with a primary antibody at 4 &#176;C O/N. The range for primary antibody dilution is normally between 1:200 and 1:5,000, determine the optimal dilution for each antibody experimentally. The dilution of antibodies used in this protocol can be found in &#8220;Table of specific reagents and equipment&#8221;.
	<ol>
		<li>To detect splice isoforms, use specific antibodies that recognize a particular isoform. Alternatively, use an antibody that recognizes the epitope in the constitutive exon coding region and detect splice isoforms simultaneously based on their different protein sizes.</li>
		<li>At the same time, monitor EMT by immunoblotting for EMT markers. Use E-cadherin, &#947;-catenin, and occludin as epithelial markers, and fibronectin, N-cadherin and vimentin as mesenchymal markers.</li>
	</ol>
	</li>
	<li>Wash membrane three times&#160;with TBS-T (50 mM Tris, 150 mM NaCl, 0.05% Tween 20, pH 7.4) at a 5-min&#160;interval.</li>
	<li>Incubate membrane with an HRP-conjugated secondary antibody in a 1:10,000 dilution for 1 hr at RT.</li>
	<li>Wash membrane three times&#160;with TBS-T at a 5-min&#160;interval.</li>
	<li>Visualize protein with a chemiluminescence detection system and expose the membrane to an autoradiography film.</li>
</ol>

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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.

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 confirmed using various EMT markers before any conclusions on changes in alternative splicing can be drawn. Furthermore, in addition to the Twist-ER-inducible EMT system described here, other EMT systems such as those induced by Snail-ER or TGF&#946; are recommended for validation of experimental findings11,30.
The switching of splice isoforms during EMT can be detected in both RNA and protein levels. Splice isoform-specific antibodies are preferred for protein analysis. When isoform-specific antibodies are not available, protein levels of splice isoforms can be determined based on their molecular weight on SDS-PAGE by immunoblotting. The RNA level of each isoform can be quantified by isoform-specific primers. Using qRT-PCR assays, the fold change of each isoform can be sensitively and precisely determined. In particular, when experimental materials are limited, such as analyzing the expression of a particular splice isoform in clinical specimens, qRT-PCR analysis provides a quantitative measure that would compensate for the lack of isoform-specific antibodies for immunohistochemistry.
The method mentioned here is suitable for the detection of known splice isoform changes during EMT. Additionally, this method can be expanded for the study of genome-wide alternative splicing and for the identification of novel splice isoform switching during EMT. To accomplish this, use of a large-scale technique, such as RNA deep-Sequencing or splicing sensitive microarray platforms, could be performed using RNAs isolated from the inducible EMT model described above. Nevertheless, qRT-PCR validation of isoform changes is required following any large-scale analysis.
In conclusion, alternative splicing is dynamically regulated during EMT, a process that is critical for embryonic development and tumor metastasis. Considering the high frequency of alternative splicing in the human genome, it is likely that the regulation of alternative splicing plays an important role in many other biological and pathological processes including cell differentiation, tissue development, and programmed cell death35-41. The protocol described herein for the detection of splice isoforms will be applicable to these other systems as well.
Additional experimental modalities, such as splicing reporter minigene assays, can be used to further investigate the regulatory mechanisms of cis-acting elements and trans-acting factors that control alternative splicing30,42,43. Furthermore, the functional role of a particular splice isoform during EMT can be investigated by silencing or ectopically expressing the specific isoform and monitoring EMT induction in the described EMT-inducible system11.

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><tbody><tr><td>4-hydroxytamoxifen</td><td>Sigma</td><td>H7904</td><td>Make stock solution by dissolving 4-hydroxytamoxifen in ethanol to 200 &amp;#956;M. Keep the solution at -20 &amp;#176;C protected from light.</td></tr><tr><td>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><td>GoScript Reverse Transcription System</td><td>Promega</td><td>A5001</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>GoTaq qPCR Master Mix</td><td>Promega</td><td>A6002</td><td>Reagent for qRT-PCR assay</td></tr><tr><td>LightCycler 480 Real-Time PCR System</td><td>Roche</td><td></td><td>Equipment for qRT-PCR assay</td></tr><tr><td>CD44 antibody</td><td>R&amp;amp;D Systems</td><td>BBA10</td><td>1:1,000 dilution</td></tr><tr><td>E-cadherin antibody</td><td>Cell Signaling Technology</td><td>4065</td><td>1:2,500 dilution for immunoblotting; 1:50 dilution for immunofluorescence</td></tr><tr><td>&amp;gamma;-Catenin antibody</td><td>Cell Signaling Technology</td><td>2309</td><td>1:1,000 dilution</td></tr><tr><td>Occludin antibody</td><td>Santa Cruz Biotechnology Inc.</td><td>sc-5562</td><td>1:500 dilution</td></tr><tr><td>Fibronectin antibody</td><td>BD Transduction Laboratories</td><td>610077</td><td>1:5,000 dilution</td></tr><tr><td>N-cadherin antibody</td><td>BD Transduction Laboratories</td><td>610920</td><td>1:2,000 dilution</td></tr><tr><td>Vimentin antibody</td><td>NeoMarkers</td><td>MS-129-p1</td><td>1:500 dilution</td></tr><tr><td>GAPDH antibody</td><td>Millipore Corporation</td><td>MAB374</td><td>1:10,000 dilution</td></tr><tr><td>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 mesenchymal markers fibronectin, N-cadherin, and vimentin (Figure 2B). Furthermore, EMT was assessed by the loss of E-cadherin localization at cell-cell junctions (Figure 2C).
The switched expression of CD44 splice isoforms during EMT was examined by qRT-PCR and immunoblotting. The human CD44 gene is comprised of nine variable exons. Alternative splicing of CD44 allows for cells to produce CD44 variants (CD44v) that include at least one variable exon, and CD44 standard (CD44s) that is devoid of all variable exons. Figure 3A depicts the strategy of primer design for the detection of various CD44 splice isoforms. For the detection of the exon-skipped product CD44s, the forward primer is located in the constitutive exon 5, while the reverse primer spans across the junction between constitutive exons 5 and 6. For the detection of CD44v splice isoforms containing the v5 and v6 variable exons, the forward and reverse primers are designed within the v5 and v6, respectively. Primers for detection of other CD44 variable exon-containing isoforms can be designed using the same strategy. Additionally, total CD44 transcript is detected using forward and reverse primers located in the constitutive exon 2 and exon 3, respectively (Figure 3A). As shown in Figure 3B, qRT-PCR analyses utilizing these primer sets indicated a significant decrease in CD44v mRNA and increase in CD44s mRNA after 14 days of TAM treatment in the Twist-ER-expressing HMLE cells. By contrast, the total transcript of CD44 remained unchanged during EMT (Figure 3C). Consistent with the results of qRT-PCR, the protein level of CD44v declined remarkably, whereas expression of the CD44s protein was greatly upregulated (Figure 3D). Therefore, the major isoform of CD44 was shifted from CD44v to CD44s during the EMT process.
<img alt="Figure 1" fo:content-width="3.5in" src="/files/ftp_upload/51845/51845fig1highres.jpg" width="600" /> 
Figure 1.&#160;Primer design. Schematic diagrams illustrating the location of primers for detecting splicing isoforms in an exon-skipping model. The gray boxes represent constitutive exons, the orange boxes represent variable exons, and the thin lines connecting boxes denote introns. The primer locations are indicated by arrows: black arrows indicate primer sets for detecting total mRNA, orange arrows indicate primer sets for amplifying exon-included isoforms, and blue arrows indicate primer sets for detecting exon-skipping isoforms.
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/51845/51845fig2highres.jpg" width="600" /> 
Figure 2.&#160;Induction of EMT. (A) Phase contrast images (10X) illustrating morphological changes in HMLE/Twist-ER cells before (untreated) and after 14 days of TAM treatment. (B) Immunoblot analysis of EMT markers in HMLE/Twist-ER cells before (untreated) and after 14 days of TAM treatment. Upon TAM treatment, epithelial markers E-cadherin, &#947;-catenin, and occludin were downregulated, and mesenchymal markers fibronectin, N-cadherin, and vimentin were upregulated. (C) Immune fluorescence images (63X) indicating the loss of E-cadherin localization at cell-cell junctions after 14 days of TAM treatment. Green staining indicates E-cadherin, and DAPI staining (blue) indicates nuclei. Scale bar =&#160;10 &#956;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/51845/51845fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" fo:content-width="5in" src="/files/ftp_upload/51845/51845fig3highres.jpg" width="500" /> 
Figure 3.&#160;Switch of CD44 splice isoforms during EMT. (A) Primer design for the detection of CD44 splice isoforms. The gray boxes represent constitutive exons, the orange boxes represent variable exons, and the thin lines connecting boxes denote introns. The primer locations are indicated by arrows: black arrows indicate primer sets for detecting CD44 total mRNA, orange arrows indicate primer sets for amplifying CD44v5-6, and blue arrows indicate primer sets for detecting CD44s. (B,&#160;C) qRT-PCR analysis of the levels of CD44 isoforms during EMT using primers that specifically detect CD44v containing variable exons v5 and v6 (v5/6) and CD44s (B), and CD44 total mRNA (C). Relative expression levels of mRNA in Day 14 cells were normalized to corresponding day 0 cells in TAM treated and non-treated groups. Error bars indicate SEM; n = 3. (D) Immunoblot analysis of CD44 isoforms during TAM-induced EMT in HMLE/Twist-ER cells. Levels of E-cadherin and N-cadherin were monitored to identify EMT status.

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.

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

Nanyang Technological University

Research

JoVE Journal

Biology

12.9K Views.  Northwestern University Feinberg School of Medicine. Alternative splicing regulation has been shown to contribute to the epithelial-mesenchymal transition (EMT), an essential cellular program in various physiological and pathological processes. Here we describe a method utilizing an inducible EMT model for the detection of alternative splicing during EMT.

Video: Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance can be caused by a range of mechanisms, including increased drug elimination, decreased drug uptake, drug inactivation and alterations of drug targets. Recent data showed that other than by well-known genetic (mutation, amplification) and epigenetic (DNA hypermethylation, histone post-translational modification) modifications, drug resistance mechanisms might also be regulated by splicing aberrations. This is a rapidly growing field of investigation that deserves future attention in order to plan more effective therapeutic approaches. The protocol described in this paper is aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of several in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes. In particular, we evaluated the differential splicing of DDX5 and PKM transcripts. The aberrant splicing detected by the computational tool MATS was validated in leukemic cells, showing that different DDX5 splice variants are expressed in the parental vs. resistant cells. In these cells, we also observed a higher PKM2/PKM1 ratio, which was not detected in the Panc-1 gemcitabine-resistant counterpart compared to parental Panc-1 cells, suggesting a different mechanism of drug-resistance induced by gemcitabine exposure.

Using RNA-sequencing to Detect Novel Splice Variants Related to Drug Resistance in In Vitro Cancer Models

Here we describe a protocol aimed at investigating the impact of aberrant splicing on drug resistance in solid tumors and hematological malignancies. To this goal, we analyzed the transcriptomic profiles of parental and resistant in vitro models through RNA-seq and established a qRT-PCR based method to validate candidate genes.

Drug resistance remains a major problem in the treatment of cancer for both hematological malignancies and solid tumors. Intrinsic or acquired resistance ...

Cancer Research

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genetics

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic plasticity drives invasion, dissemination and metastasis. Indeed, while most of the studies of phenotypic plasticity have been in the context of epithelial-derived carcinomas, it turns out sarcomas, which are mesenchymal in origin, also exhibit phenotypic plasticity, with a subset of sarcomas undergoing a phenomenon that resembles a mesenchymal-epithelial transition (MET). Here, we developed a method comprising the miR-200 family and grainyhead-like 2 (GRHL2) to mimic this MET-like phenomenon observed in sarcoma patient samples.We sequentially express GRHL2 and the miR-200 family using cell transduction and transfection, respectively, to better understand the molecular underpinnings of these phenotypic transitions in sarcoma cells. Sarcoma cells expressing miR-200s and GRHL2 demonstrated enhanced epithelial characteristics in cell morphology and alteration of epithelial and mesenchymal biomarkers. Future studies using these methods can be used to better understand the phenotypic consequences of MET-like processes on sarcoma cells, such as migration, invasion, metastatic propensity, and therapy resistance.

We present here a cell culture method for inducing mesenchymal-epithelial transitions (MET) in sarcoma cells based on combined ectopic expression of microRNA-200 family members and grainyhead-like 2 (GRHL2). This method is suitable for better understanding the biological impact of phenotypic plasticity on cancer aggressiveness and treatments.

Phenotypic plasticity refers to a phenomenon in which cells transiently gain traits of another lineage. During carcinoma progression, phenotypic ...

Developmental Biology

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of ...

Using the E1A Minigene Tool to Study mRNA Splicing Changes

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides constitutive splicing, alternative mRNA splicing allows the expression of multifunctional proteins from one gene. As interactome studies are generally the first analysis for new or unknown proteins, the association of the bait protein with splicing factors is an indication that it can participate in mRNA splicing process, but to determine in what context or what genes are regulated is an empirical process. A good starting point to evaluate this function is using the classical minigene tool. Here we present the adenoviral E1A minigene usage for evaluating the alternative splicing changes after different cellular stress stimuli. We evaluated the splicing of E1A minigene in HEK293 stably overexpressing Nek4 protein after different stressing treatments. The protocol includes E1A minigene transfection, cell treatment, RNA extraction and cDNA synthesis, followed by PCR and gel analysis and quantification of the E1A spliced variants. The use of this simple and well-established method combined with specific treatments is a reliable starting point to shed light on cellular processes or what genes can be regulated by mRNA splicing.

This protocol presents a rapid and useful tool for evaluating the role of a protein with uncharacterized function in alternative splicing regulation after chemotherapeutic treatment.

mRNA processing involves multiple simultaneous steps to prepare mRNA for translation, such as 5&#180;capping, poly-A addition and splicing. Besides ...

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5&#8242; splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5&#8242; region that basepairs with the 5&#8242;-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5&#8242; splice-sites. Insertion of cis-acting&#160;regulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5&#8242; splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

This protocol describes a minigene reporter assay to monitor the impact of 5&#180;-splice site mutations on splicing and develops suppressor U1 snRNA for the rescue of mutation-induced splicing inhibition. The reporter and suppressor U1 snRNA constructs are expressed in HeLa cells, and splicing is analyzed by primer extension or RT-PCR.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes ...

Biochemistry

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.

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

Identification of Alternative Splicing and Polyadenylation in RNA-seq Data

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also be utilized to explore other complex regulatory mechanisms at the exon level. Alternative splicing and polyadenylation play a crucial role in the functional diversity of a gene by generating different isoforms to regulate gene expression at the post-transcriptional level, and limiting analyses to the whole gene level can miss this important regulatory layer. Here, we demonstrate detailed step by step analyses for identification and visualization of differential exon and polyadenylation site usage across conditions, using Bioconductor and other packages and functions, including DEXSeq, diffSplice from the Limma package, and rMATS.

Alternative splicing (AS) and alternative polyadenylation (APA) expand the diversity of transcript isoforms and their products. Here, we describe bioinformatic protocols to analyze bulk RNA-seq and 3' end sequencing assays to detect and visualize AS and APA varying across experimental conditions.

As well as the typical analysis of RNA-Seq to measure differential gene expression (DGE) across experimental/biological conditions, RNA-seq data can also ...

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded together.
Splicing is Mediated by the Spliceosome
Splicing occurs in the nucleosome and is mediated by a complex of proteins and small RNAs called small nuclear ribonucleoproteins (snRNPs). snRNPs, together with other proteins, form the spliceosome, which recognizes specific nucleotide sequences at the ends of the exon and intron. First, it binds to a GU-containing sequence at the 5&#39; end of the intron and to a branch point sequence containing an A towards the 3&#39; end of the intron. In a number of carefully-orchestrated steps, other snRNPs then bring the branch point close to the 5&#39; splice site. Subsequently, a chemical reaction cleaves the 5&#39; end of the intron from its upstream exon and attaches it to the branch point, forming a loop called a lariat. To release the lariat, the AG-containing sequence of the intron near the 5&#39; end of the downstream exon reacts with the 3&#39; end of the upstream exon. This reaction patches the two exons together, concluding the splicing process.
Splicing Allows the Expression of Several Proteins from a Single Gene
Typically, exons are joined together in the order in which they appear in a gene. However, during alternative splicing, different combinations of exons in pre-mRNA are combined to form mature mRNA. This produces several different proteins from a single pre-mRNA transcript.
Different patterns of alternative splicing include exon skipping, alternative 5&#39; or 3&#39; splice sites, and intron retention. These patterns are guided by the length of exons or introns and the strength of the splicing signal at the splice sites. Because of this, exons that are shorter than other exons may be overlooked by the spliceosome and omitted from the mature mRNA. In contrast, introns that are significantly shorter than other introns may evade removal by the spliceosome and are retained in the mature mRNA. As a result, alternative splicing generates variants of mature mRNA that were copied from the same stretch of DNA. The RNA sequence variants produce different proteins with additional or fewer amino acids, shifts in the reading frame, or a premature stop codon. These protein isoforms have different biological properties, including function, cellular localization, and interaction with other proteins, thereby playing a vital role in tissue- and environment-specific gene expression.
Abnormal Splicing Can Cause Diseases
Errors in splicing can produce aberrant protein isoforms, which may contribute to diseases, including cancer. For instance, alternative splicing of the BCL2L1 gene generates a long and short protein isoform&#8212;BCL-XL and BCL-XS, respectively&#8212; through the use of alternative 5&#39; splice sites. The longer BCL-XL isoform promotes cell survival and is highly expressed in several types of cancers (e.g., blood, breast, and liver cancers). Expression of the short BCL-XS isoform that promotes cell death is suppressed in cancer.

RNA Splicing, Lariat and the Spliceososme

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called ...

Detection of Alternative Splicing During Epithelial-Mesenchymal Transition

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RNA Splicing