The authors would like to acknowledge Julia Hatler and Martin Ramsden (R &amp; D Systems) for helpful discussion and manuscript review.

1. Induction of EMT
<ol>
	<li>Warm culture media to 37 &deg;C in a water bath. The culture media used is the same as the media used for standard culture of your cells of interest. For example, the A549 human lung carcinoma cell line is grown in Kaighn's F12 supplemented with 10% fetal bovine serum and 2mM glutamine.</li>
	<li>Harvest cells of interest using a dissociation solution (e.g. TrypLE Express).</li>
	<li>Suspend cells in pre-warmed culture media in a 15 ml conical tube.</li>
	<li>Centrifuge the cell suspension at 400 x g for 5 min.</li>
	<li>Carefully remove the supernatant by pouring into a waste container.</li>
	<li>Suspend the cell pellet in pre-warmed culture media.</li>
	<li>Count viable cells using Trypan Blue. Remove a sample of the cell suspension and dilute in 0.4% Trypan Blue solution. Place 10 &mu;l of the diluted sample on a hemocytometer and count viable cells (cells that do not turn blue). Use cell counts to determine the cell density in your solution.</li>
	<li>Plate cells onto tissue culture treated plates or flasks at 0.9-1.0 x 104 cells per cm2. For example, 0.5 x 106 cells plated in a 100 mm plate in culture media containing 1X EMT Inducing Media Supplement (6 ml media per 100 mm plate).</li>
	<li>Culture plated cells in a 37 &deg;C/5% CO2 incubator. Monitor cell morphology daily.</li>
	<li>Three days after plating, remove media and replace with fresh pre-warmed culture media containing 1X EMT Inducing Media Supplement. Continue to culture in a 37 &deg;C/ 5% CO2 incubator.</li>
	<li>Five days after plating, cells are ready for analysis. Cell morphology is visualized by inverted light microscopy.</li>
</ol>
2. Analysis of Protein Expression by Immunocytochemistry
<ol>
	<li>Prepare sterile 12 mm coverslips for a 24-well plate by placing coverslips in a petri dish containing 95% ethanol. Gently remove coverslips from ethanol using a needle and curved forceps and flame sterilize. Place sterile coverslip into one well of a 24-well plate. Repeat with remaining coverslips.</li>
	<li>Prepare the cell suspension as described in section 1.1 - 1.7.</li>
	<li>Plate 1.6 x 104 cells / well in 0.5 ml pre-warmed culture media containing 1X EMT Inducing Media Supplement.</li>
	<li>Grow and feed cells as described in sections 1.9 - 1.11.</li>
	<li>Five days after plating, remove media and fix cells with 300 &mu;l/well of 4% paraformaldehyde in 1X phosphate buffered saline (PBS) for 20 min at room temperature.</li>
	<li>Remove fixative and rinse cells 2x with 1X PBS, 500 &mu;l/well.</li>
	<li>Incubate cells in 400 &mu;l/well of blocking buffer (1X PBS containing 1% bovine serum albumin (BSA), 10% normal donkey serum, and 0.3% Triton X-100) for 1 hr at room temperature.</li>
	<li>Incubate in primary antibody at manufacturers recommended concentration in 400 &mu;l/well of blocking buffer for 3 hr at room temperature or overnight at 4 &deg;C. When using a primary antibody directly conjugated to a fluorochrome, incubate samples in the dark.</li>
	<li>Wash cells three times in 500 &mu;l/well of 1X PBS containing 0.1% BSA for 5 min each wash. When using a primary antibody directly conjugated to a fluorochrome, proceed directly to step 2.12.</li>
	<li>Incubate cells in secondary antibody at manufacturer's recommended concentration in 400 &mu;l/well of 1X PBS containing 1% BSA for 1 hr at room temperature in the dark.</li>
	<li>Wash cells three times in 500 &mu;l/well of 1X PBS containing 0.1% BSA for 5 min each wash.</li>
	<li>If desired, counterstain nuclei with DAPI solution.</li>
	<li>Rinse cells in deionized water and mount coverslips face down on a slide using mounting media.</li>
</ol>

<ol>
	<li>Kalluri, R., 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=The+basics+of+epithelial-mesenchymal+transition.">The basics of epithelial-mesenchymal transition.</a> Journal of Clinical Investigation. 119, 1420-1428 (2009).</li><li>Thiery, 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=Epithelial-mesenchymal+transitions+in+tumour+progression.">Epithelial-mesenchymal transitions in tumour progression.</a> Nature Reviews Cancer. 2, 442-454 (2002).</li><li>Heuberger, J., Birchmeier, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+of+cadherin-mediated+cell+adhesion+and+canonical+Wnt+signaling.">Interplay of cadherin-mediated cell adhesion and canonical Wnt signaling.</a> Cold Spring Harbor Perspectives in Biology. 2, 1-23 (2010).</li><li>Onder, T. T. <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+E-cadherin+promotes+metastasis+via+multiple+downstream+transcriptional+pathways.">Loss of E-cadherin promotes metastasis via multiple downstream transcriptional pathways.</a> Cancer Research. 68, 3645-3654 (2008).</li><li>Tian, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=E-cadherin/&#946;-catenin+complex+and+the+epithelial+barrier.">E-cadherin/&#946;-catenin complex and the epithelial barrier.</a> Journal of Biomedicine and Biotechnology. 2011, 1-6 (2011).</li><li>Scheel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paracrine+and+autocrine+signals+induce+and+maintain+mesenchymal+and+stem+cell+states+in+the+breast.">Paracrine and autocrine signals induce and maintain mesenchymal and stem cell states in the breast.</a> Cell. 145, 926-940 (2011).</li><li>Xu, J., Lamouille, S., Derynck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;-induced+epithelial+to+mesenchymal+transition.">TGF-&#946;-induced epithelial to mesenchymal transition.</a> Cell Research. 19, 156-172 (2009).</li><li>Kasai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-&#946;1+induces+human+alveolar+epithelial+to+mesenchymal+cell+transition.">TGF-&#946;1 induces human alveolar epithelial to mesenchymal cell transition.</a> EMT). Respiratory Research. 6, 56-70 (2005).</li><li>Miettinen, P. 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=TGF-&#946;+induced+transdifferentiation+of+mammary+epithelial+cells+to+mesenchymal+cells:+involvement+of+type+I+receptors.">TGF-&#946; induced transdifferentiation of mammary epithelial cells to mesenchymal cells: involvement of type I receptors.</a> Journal of Cell Biology. 127, 2021-2036 (1994).</li><li>Pelton, R. W., 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=Differential+expression+of+genes+encoding+TGFs+&#946;1,+&#946;2,+and+&#946;3+during+murine+palate+formation.">Differential expression of genes encoding TGFs &#946;1, &#946;2, and &#946;3 during murine palate formation.</a> Dev. Biol. 141, 456-460 (1990).</li><li>Taube, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Core+epithelial-to-mesenchymal+transition+interactome+gene-expression+signature+is+associated+with+claudin-low+and+metaplastic+breast+cancer+subtypes.">Core epithelial-to-mesenchymal transition interactome gene-expression signature is associated with claudin-low and metaplastic breast cancer subtypes.</a> PNAS. 107, 15449-15454 (2010).</li><li>Roussos, 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=AACR+special+conference+on+epithelial-mesenchymal+transition+and+cancer+progression+and+treatment.">AACR special conference on epithelial-mesenchymal transition and cancer progression and treatment.</a> Cancer Research. 70, 7360-7364 (2010).</li><li>Polyak, K., 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=Transitions+between+epithelial+and+mesenchymal+states:+acquisition+of+malignant+and+stem+cell+traits.">Transitions between epithelial and mesenchymal states: acquisition of malignant and stem cell traits.</a> Nature Reviews Cancer. 9, 265-273 (2009).</li><li>Brown, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+by+transforming+growth+factor-&#946;1+of+epithelial+to+mesenchymal+transition+is+a+rare+event+in+vitro.">Induction by transforming growth factor-&#946;1 of epithelial to mesenchymal transition is a rare event in vitro.</a> Breast Cancer Research. 6, 215-231 (2004).</li><li>De Falco, 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=CD44+proteolysis+increases+CREB+phosphorylation+and+sustains+proliferation+of+thyroid+cancer+cells.">CD44 proteolysis increases CREB phosphorylation and sustains proliferation of thyroid cancer cells.</a> Cancer Research. 72, 1449-1458 (2012).</li><li>Xie, L. <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+the+Erk+pathway+is+required+for+TGF-&#946;1-induced+EMT+in+vitro.">Activation of the Erk pathway is required for TGF-&#946;1-induced EMT in vitro.</a> Neoplasia. 6, 603-610 (2004).</li><li>Bachelder, R. 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=Glycogen+synthase+kinase-3+is+an+endogenous+inhibitor+of+Snail+transcription:+implication+for+the+epithelial-mesenchymal+transition.">Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implication for the epithelial-mesenchymal transition.</a> Journal of Cell Biology. 168, 29-33 (2005).</li><li>Pon, Y. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p70+S6+kinase+promotes+epithelial+to+mesenchymal+transition+through+Snail+induction+in+ovarian+cancer+cells.">p70 S6 kinase promotes epithelial to mesenchymal transition through Snail induction in ovarian cancer cells.</a> Cancer Research. 68, 6524-6532 (2008).</li><li>Wegner, G. 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=Profiling+changes+in+receptor+tyrosine+kinase+phosphorylation+using+antibody+arrays.">Profiling changes in receptor tyrosine kinase phosphorylation using antibody arrays.</a> J. Vis. Exp. , (2012).</li></ol>

R &amp; D Systems, Inc. is a for-profit, public company. Production and Free Access to this article is sponsored by R &amp; D Systems, Inc.

Previous methods for EMT induction have generally used TGF-&beta; stimulation or genetic modification. Although TGF-&beta; alone has been shown to induce EMT in some cell types, it has now been demonstrated that TGF-&beta; is necessary for EMT, but it is not sufficient in all cell types6,14. Using genetic modification for EMT induction is time consuming and labor intensive. The method described here uses a combination of factors including, anti-human E-cadherin, anti-human sFRP-1, anti-human Dkk-1, recombinant human Wnt-5a, and recombinant human TGF-&beta;1 to reliably induce EMT. This combination of factors is designed to block E-cadherin-based adhesion, a critical step for EMT induction4, and enhance Wnt signaling by adding the Wnt-5a protein as well as using blocking antibodies to the Wnt inhibitors, sFRP-1 and Dkk-1. Wnt and TGF-&beta; signaling have been shown to act in an autocrine loop to induce and maintain the mesenchymal cell state6. This induction method avoids genetic manipulation and is more effective than using TGF-&beta; alone. Importantly, we are able to observe induction of EMT in cell types, including MCF7 and PANC-1 cells, that have been previously shown not to respond solely to TGF-&beta; stimulation (Figure 5)14.
Cells treated with the EMT Inducing Media Supplement show less compact and increased spindle-shaped morphology (Figure 1). Additionally, there is a decrease in surface E-cadherin expression concurrent with an increase in Fibronectin expression, which are hallmarks of EMT (Figures 2 and 3). Additional mesenchymal markers such as Vimentin and Snail are also upregulated in the EMT induced cells as compared to uninduced controls (Figure 4). Increased migration and invasion abilities are the main functional characteristics of mesenchymal cells. In in vitro migration and invasion assays, cells treated with the EMT Inducing Media Supplement demonstrated a significantly increased capacity to migrate and invade (Figure 6).
This simple method for EMT induction is useful for the study of EMT signaling as demonstrated using the antibody array expression data shown in Figure 7. Different cell types can be compared pre- and post-EMT induction to obtain common expression signatures associated with EMT, such as the increase in phosphorylated CREB and ERK1/2 seen in both MCF7 and A549 EMT-induced cells. S133 phosphorylation of CREB stimulates DNA binding and has been shown to act downstream of TGF-&beta;1-induced EMT15. ERK1/2 phosphorylation has also been shown to act downstream of TGF-&beta;1-induced EMT16. Differences in signaling pathways among cells types can also be elucidated as seen with the increase in GSK-3&beta; phosphorylation in A549 cells versus p70S6K phosphorylation in MCF7. GSK-3&beta; phosphorylation at serine 9 decreases it function, and GSK-3&beta; has been shown to inhibit the pro-EMT transcription factor, Snail17. In contrast, p70S6K induces Snail activity and its phosphorylation is associated with activation of this protein18.
Overall, straightforward and reliable induction of EMT, particularly in cells previously shown not to respond solely to TGF-&beta;, is useful for understanding the biology of EMT, identifying common expression signatures for use as prognostic markers, and developing and evaluating novel therapeutics for cancer and fibrotic disease.

Epithelial to mesenchymal transition (EMT) is the process by which a polarized epithelial cell undergoes a variety of changes resulting in a highly motile, fibroblast-like mesenchymal cell. This process occurs, in part, through gene expression changes and the degradation of adherens junctions, resulting in an increased ability to migrate and invade. Physiologically, EMT plays important roles during embryonic development and wound healing. Loss of proper control over EMT can lead to fibrosis and metastasis of carcinomas1,2. 
	The reduction of E-cadherin on the surface of epithelial cells is a critical step in the progression of a cell through EMT3,4. E-cadherin is a single-pass transmembrane protein that directly interacts with cadherin proteins on adjacent cells. In addition to its role in cell adhesion, E-cadherin influences cell signaling via interactions between the cytoplasmic tail of E-cadherin and a variety of regulatory proteins, notably &beta;-catenin. &beta;-catenin plays a role in the stabilization of adherens junctions. During canonical Wnt signaling, &beta;-catenin is released from E-cadherin and translocated to the nucleus where it acts as a downstream transcription factor in the Wnt pathway3,4,5. In the nucleus, &beta;-catenin has been shown to increase the transcription of several EMT-related factors including Twist, Slug, Fibronectin, and a variety of matrix metalloproteases3,6.
	In addition to Wnts, TGF-&beta; signaling has been shown to play a significant role in EMT induction both during normal development and in diseased states7,8,9. TGF-&beta; is involved in the EMT that occurs during palate formation and in the developing heart10. It has also been implicated in fibrosis and in cancer progression to metastasis7.
	The procedure described here provides consistent induction of EMT in a variety of cell types without the need for genetic manipulation. This procedure uses a cocktail of E-cadherin, sFRP-1, and Dkk-1 blocking antibodies and Wnt-5a and TGF-&beta;1 recombinant proteins. This cocktail is designed to block E-cadherin-based adhesion while enhancing Wnt and TGF-&beta; signaling. The ability for robust EMT induction is critical for understanding the biology of this process and how to manipulate it for therapeutic purposes. Current common markers of EMT, E-cadherin (downregulated in EMT) and Vimentin (upregulated in EMT), can be found co-expressed in cancers and thus do not provide the best diagnostic markers of potential metastatic cells12. Importantly, the analysis of a variety of cell types that have undergone EMT is useful to develop common gene expression signatures that can be used for diagnostic purposes in cancer and fibrosis11. Additionally, recent studies have demonstrated that EMT may play a role in the formation of cancer stem cells, suggesting that cells that have undergone EMT may be useful for drug screening to identify compounds that can target these cells13.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		 </tr>
		 <tr>
			<td>Epithelial cells of interest</td>
			<td></td>
			<td></td>
			<td>We have tested: A431, A549, HT29, T98G, MCF10A, MCF7, and PANC1</td>
		 </tr>
		 <tr>
			<td>EMT Inducing Media Supplement</td>
			<td>R &amp; D Systems</td>
			<td>CCM017</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Recombinant Human TGF-&beta;1</td>
			<td>R &amp; D Systems</td>
			<td>240-B</td>
			<td>Used at 10ng/ml</td>
		 </tr>
		 <tr>
			<td>Anti-E-Cadherin NL557 conjugated antibody</td>
			<td>R &amp; D Systems</td>
			<td>NL648R</td>
			<td>Used at a 1:10 dilution for immunocytochemistry</td>
		 </tr>
		 <tr>
			<td>Anti-E-Cadherin </td>
			<td>R &amp; D Systems</td>
			<td>MAB1838</td>
			<td>Used at 0.1 &mu;g/ml for western blotting</td>
		 </tr>
		 <tr>
			<td>Anti-Fibronectin</td>
			<td>R &amp; D Systems</td>
			<td>MAB1918</td>
			<td>Used at 10 &mu;g/ml for immunocytochemistry; Used at 0.5 &mu;g/ml for western blotting</td>
		 </tr>
		 <tr>
			<td>Anti-GAPDH</td>
			<td>R &amp; D Systems</td>
			<td>AF5718</td>
			<td>Used at 0.2 &mu;g/ml for western blotting</td>
		 </tr>
		 <tr>
			<td>Goat Anti-Mouse NL493 Secondary Antibody</td>
			<td>R &amp; D Systems</td>
			<td>NL009</td>
			<td>Used at a 1:200 dilution for immunocytochemistry</td>
		 </tr>
		 <tr>
			<td>Donkey Anti-Goat HRP Secondary Antibody</td>
			<td>R &amp; D Systems</td>
			<td>HAF109</td>
			<td>Used at 1:2000 for western blotting</td>
		 </tr>
		 <tr>
			<td>Goat Anti-Mouse HRP Secondary Antibody</td>
			<td>R &amp; D Systems</td>
			<td>HAF007</td>
			<td>Used at 1:2000 for western blotting</td>
		 </tr>
		 <tr>
			<td>EMT 3-Color Immunocytochemistry Kit</td>
			<td>R &amp; D Systems</td>
			<td>SC026</td>
			<td>Used according to the manufacturer's instructions. This kit contains directly conjugated antibodies against E-cadherin, Vimentin, and Snail.</td>
		 </tr>
		 <tr>
			<td>96 Well BME Cell Invasion Assay</td>
			<td>R &amp; D Systems</td>
			<td>3455-096-K</td>
			<td>This kit was used according to the manufacturer's recommendations.</td>
		 </tr>
		 <tr>
			<td>Proteome Profiler Human Phospho-MAPK Array Kit</td>
			<td>R &amp; D Systems</td>
			<td>ARY002B</td>
			<td>This kit was used according to the manufacturer's recommendations19.</td>
		 </tr>
		 <tr>
			<td>Mounting Media</td>
			<td>R &amp; D Systems</td>
			<td>CTS011</td>
			<td></td>
		 </tr>
		 <tr>
			<td>0.4% Trypan Blue Solution</td>
			<td>Life Technologies</td>
			<td>15350-061</td>
			<td></td>
		 </tr>
		 <tr>
			<td>1X Phosphate Buffered Saline (PBS)</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>95% Ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A3311</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Normal Donkey Serum</td>
			<td>Jackson Labs</td>
			<td>017-000-121</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Triton-X-100</td>
			<td>Roche Molecular Biochemicals</td>
			<td>789-704</td>
			<td>Dilute in 1X PBS to make a 10% stock solution</td>
		 </tr>
		 <tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
			<td>Dilute in 1X PBS to a 4% working concentration.</td>
		 </tr>
		 <tr>
			<td>DAPI Solution</td>
			<td>Sigma</td>
			<td>D9542</td>
			<td>Make stock solution at 14.3mM by dissolving in water. Working solution is a 1:5000 dilution of the stock solution in 1X PBS.</td>
		 </tr>
		 <tr>
			<td>Fine pointed curved forceps</td>
			<td>Thermo-Fisher Scientific</td>
			<td>08-875</td>
			<td></td>
		 </tr>
		 <tr>
			<td>12 mm coverslips</td>
			<td>Carolina Biological</td>
			<td>633009</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Nonfat dry milk</td>
			<td></td>
			<td></td>
			<td>Used at 5% in TBST for blocking in western blotting</td>
		 </tr>
		 <tr>
			<td>TBST [25mM Tris, pH 7.4, 0.15M NaCl, 0.1% Tween 20]</td>
			<td></td>
			<td></td>
			<td>For western blotting blocking and washing</td>
		 </tr>
		 <tr>
			<td>PVDF Membrane</td>
			<td></td>
			<td></td>
			<td>For western blotting </td>
		 </tr>
		 <tr>
			<td>4-20% SDS-page gel</td>
			<td></td>
			<td></td>
			<td>For western blotting</td>
		 </tr>
		 <tr>
			<td>ECL substrate</td>
			<td></td>
			<td></td>
			<td>For western blotting</td>
		 </tr>
		 <tr>
			<td>15 ml conical tubes</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Plates/flasks, tissue culture treated</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>24-well plates, tissue culture treated</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipettes / pipette tips</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Pipet Aid / pipets</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Hemocytometer</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>37 &deg;C Water Bath</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>37 &deg;C/5%CO2 Incubator</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Inverted light microscope</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		 <tr>
			<td>Fluorescence microscope</td>
			<td></td>
			<td></td>
			<td></td>
		 </tr>
		</tbody>
 </table>

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.

The EMT inducing culture conditions described here provide a robust method for the induction of EMT in a variety of cell types. Figures 1 and 2 demonstrate the morphology of and marker expression levels for 4 different human cell lines: T98G glioblastoma cells, HT29 colon adenocarcinoma cells, A549 lung carcinoma cells, and MCF10A mammary epithelial cells. Cells that were treated with the EMT Inducing Media Supplement changed from a classical epithelial morphology (Figures 1A - 1D) to a mesenchymal, spindle-shaped morphology (Figures 1E - 1H). EMT-induced cells appeared less densely packed into tight colonies compared to uninduced cells. Uninduced MCF10A samples contained tightly packed clusters surrounded by more loosely packed cells. These tightly packed clusters were E-cadherin positive (Figure 2D) and disappeared upon treatment with the EMT Inducing Media Supplement (Figure 2H).
	EMT was also assessed by the downregulation of epithelial markers and upregulation of mesenchymal markers. The downregulation of E-cadherin expression is typically observed following EMT induction in different cell types3,4. Figure 2 demonstrates surface expression of E-cadherin in the majority of untreated cells (Figures 2A - 2D; red) in comparison to its absence after EMT induction (Figures 2E - 2H; red). One cell line, T98G, was found to have an extremely low basal level of E-cadherin prior to EMT induction, which precluded its analysis. Upregulation of the mesenchymal marker, Fibronectin, was also visible after induction with the EMT Inducing Media Supplement (Figures 2A - 2D versus Figures 2E - 2H; green).
	The expression levels of E-cadherin and Fibronectin seen by immunocytochemistry were further confirmed through western blotting of total cell lysates (Figure 3). The western blot confirmed downregulation of E-cadherin and upregulation of Fibronectin expression seen by immunocytochemistry. The bands were quantitated using densitometry and normalized to GAPDH to determine relative fold change following EMT induction. E-cadherin levels were reduced 6.4 and 3.4 fold in A549 and MCF10A cells, respectively. Fibronectin levels were increased 4.6, 4.1, and 2.3 fold in T98G, A549, and MCF10A cells, respectively. Although the western blot does not show significant reduction of E-cadherin protein levels in HT29 cells post-EMT induction, the immunocytochemistry results demonstrate a reduction in surface expression of E-cadherin (Figure 2B versus 2F), which is unable to be distinguished in western blotting of total cell lysates. 
	To further evaluate EMT status, additional known markers for EMT were analyzed pre-and post-EMT induction. Consistent with previous results, E-cadherin was downregulated in all cell lines examined, indicating that EMT was induced. In addition, the mesenchymal markers, Vimentin and Snail, were upregulated in A549, T98G, and MCF10A cells following treatment with EMT Inducing Media Supplement (Figure 4). The HT29 cell line did not show expression of these mesenchymal markers either with or without EMT induction (data not shown), which may indicate that these cells utilize alternate pathways for EMT induction.
	Although TGF-&beta; is sufficient to induce EMT in certain cell contexts7,8,9, other cell types do not respond solely to the addition of TGF-&beta;14. MCF7 human breast cancer cells and PANC-1 human pancreatic carcinoma cells are both reported not to respond to TGF-&beta; signaling alone14. To determine whether both lines could undergo EMT in vitro, cells were cultured in the presence of the EMT Inducing Media Supplement. Cells were stimulated with recombinant TGF-&beta;1 alone, at the concentration within the EMT Inducing Media Supplement. These cells retained their epithelial morphology, consisting of tightly packed colonies of cells (data not shown) and surface E-cadherin levels were similar to those seen in control cells (Figures 5C and 5D versus Figures 5A and 5B). In contrast, cells stimulated with the EMT Inducing Media Supplement showed a drastic decrease in surface E-cadherin levels (Figures 5E and 5F). These cells also obtained a more mesenchymal morphology, as cells became less compact and more spindle-like (data not shown). These results demonstrate that the EMT inducing method described here is able to promote EMT morphology and marker expression changes in cells typically resistant to TGF-&beta;-induced EMT.
	In addition to changes in marker expression, another hallmark of mesenchymal cells is their ability to migrate and invade. Migration and invasion of cells cultured with or without the EMT Inducing Media Supplement were analyzed using the 96 well BME Cell Invasion Assay according to the manufacturer's instructions. Briefly, cells were seeded onto 96-well plates (50,000 cells/well) containing filter inserts with 8.0 &mu;m pores. After 48 hr, migration was assessed by calcein AM staining of cells that had migrated through the filter. Each sample was tested in triplicate twice with similar results. Representative results from one experiment are shown in Figure 6A. Statistically significant increases (P-value&lt;0.003) in cell migration were seen with both A549 and PANC-1 cells following EMT induction. To assess the ability of these cells to invade (i.e. migrate through extracellular matrix), the same assay was performed with a basement membrane extract-coated filter. EMT-induced cells showed a statistically significant (P- value&lt;0.001) increase in invasion capabilities compared to untreated cells as seen in Figure 6B.
	The robust induction of EMT is useful for the analysis of gene expression changes and signaling taking place in these cells. Antibody-based array analysis using lysates from both treated and untreated cells is one method to analyze the expression levels of a variety of molecules in a single sample. As an example, we analyzed the levels of phosphorylated MAPK family members in lysates from un-induced and EMT-induced MCF7 and A549 cells using the Proteome Profiler Human Phospho-MAPK Array Kit. The array was run twice from independent EMT-induction treatments with similar results. Figure 7 shows representative data from one array. Both cell types exhibited increased phosphorylation of CREB (S133), ERK1 (T202/Y204), and ERK2 (T185/Y187) in EMT-induced cells compared to controls. Differences in signaling events were observed between the two cell types as well. A549 cells showed an increase in GSK-3 &beta; (S9) phosphorylation, whereas MCF7 cells showed increased p70S6K (T421/S424) phosphorylation. 
	<img src="/files/ftp_upload/50478/50478fig1.jpg" alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/50478/50478fig1highres.jpg" fo:width="6in"/> 
 Figure 1. Mesenchymal morphology induction after stimulation with the EMT Inducing Media Supplement. (A - D) Epithelial morphology in the four cell types indicated following culture for 5 days in standard culture media without the EMT Inducing Media Supplement. (E - H) Mesenchymal morphology in the four cell types indicated cultured with the EMT Inducing Media Supplement for 5 days.
	<img src="/files/ftp_upload/50478/50478fig2.jpg" alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/50478/50478fig2highres.jpg" fo:width="6in"/> 
 Figure 2. Downregulation of epithelial marker expression and upregulation of mesenchymal marker expression in EMT-induced cells. Cells were stained by immunofluorescence to detect the expression of the epithelial marker, E-cadherin (red), and the mesenchymal marker, Fibronectin (green). (A - D) Control cells cultured without the EMT Inducing Media Supplement for 5 days. (E - H) Cells cultured with the EMT Inducing Media Supplement for 5 days. All cells are counterstained with DAPI (blue).
	<img src="/files/ftp_upload/50478/50478fig3.jpg" alt="Figure 3"/> 
 Figure 3. Confirmation of epithelial and mesenchymal marker expression with western blot analysis. Western blot of total cell lysates of the four cell types indicated, treated with (EMT) or without (-) the EMT Inducing Media Supplement for 5 days. Bands for E-cadherin, Fibronectin, and the GAPDH loading control are as indicated on the left. The size marker is as indicated on the right. 
	<img src="/files/ftp_upload/50478/50478fig4.jpg" alt="Figure 4"/> 
 Figure 4. Upregulation of the mesenchymal markers, Vimentin and Snail in EMT-induced cells. Cells were stained by multicolor immunofluorescence to detect expression of the epithelial marker, E-cadherin (gray), and the mesenchymal markers, Vimentin (green) and Snail (red). (A - C) Control cells cultured without the EMT Inducing Media supplement for 5 days. (D - F) Cells cultured with the EMT Inducing Media Supplement for 5 days.
	<img src="/files/ftp_upload/50478/50478fig5.jpg" alt="Figure 5" fo:content-width="4in" fo:src="/files/ftp_upload/50478/50478fig5highres.jpg" fo:width="6in"/> 
 Figure 5. Induction of EMT in TGF-&beta;1 non-responsive cells. (A and B) Cells cultured with media alone for 5 days. (C and D) Cells cultured in media containing 10 ng/ml of TGF-&beta;1 for 5 days. (E and F) Cells cultured in EMT Inducing Media Supplement for 5 days. All cells are stained for E-cadherin expression (red) and counterstained with DAPI (blue).
	<img src="/files/ftp_upload/50478/50478fig6.jpg" alt="Figure 6"/> 
 Figure 6. EMT-induced cells have increased migration and invasion capacities. A. Average number of cells that migrated through an 8 &mu;m pore filter after incubation for 48 hr. B. Average number of cells that were able to invade through an 8 &mu;m pore filter coated with basement membrane extract after incubation for 48 hr. Error bars indicate the standard deviation over 3 wells.
	<img src="/files/ftp_upload/50478/50478fig7.jpg" alt="Figure 7"/> 
 Figure 7. Antibody-based array expression analysis of EMT induction. Lysates from A549 (A and C) and MCF7 (B and D) cells cultured with or without EMT Inducing Media Supplement were used for array analysis using the Proteome Profiler Human Phospho-MAPK Array Kit. Spots highlighted in A and B were then quantitated using densitometry. Comparisons of un-induced to EMT-induced lysates are shown in the corresponding bar graphs below (C and D). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/50478/50478fig7large.jpg" target="_blank">Click here to view larger figure</a>.

Watch this Scientific Journal Video about Induction and Analysis of Epithelial to Mesenchymal Transition at JoVE.com

Induction and Analysis of Epithelial to Mesenchymal Transition

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 ...

induction-and-analysis-of-epithelial-to-mesenchymal-transition

Nanyang Technological University

Research

JoVE Journal

Biology

35.6K Views.  R&D Systems, Inc.. 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.

Video: Induction and Analysis of Epithelial to 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.

Światło / ciemność test przejścia dla Myszy

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, ...

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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.

Hair follicle morphogenesis, a complex process requiring interaction between epithelia-derived keratinocytes and the underlying mesenchyme, is an ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

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.

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 ...

Manipulating the Murine Lacrimal Gland

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for vision. In the human a single LG resides in the orbit above the lateral end of each eye delivering tears to the ocular surface through 3 - 5 ducts. The mouse has three pairs of major ocular glands, the most studied of which is the exorbital lacrimal gland (LG) located anterior and ventral to the ear. Similar to other glandular organs, the LG develops through the process of epithelial branching morphogenesis in which a single epithelial bud within a condensed mesenchyme undergoes multiple rounds of bud and duct formation to form an intricate interconnected network of secretory acini and ducts. This elaborate process has been well documented in many other epithelial organs such as the pancreas and salivary gland. However, the LG has been much less explored and the mechanisms controlling morphogenesis are poorly understood. We suspect that this under-representation as a model system is a consequence of the difficulties associated with finding, dissecting and culturing the LG. Thus, here we describe dissection techniques for harvesting embryonic and post-natal LG and methods for ex vivo culture of the tissue.

The lacrimal gland (LG) is a branching organ that produces the aqueous components of tears necessary for maintaining vision and ocular health. Here we describe murine LG dissection and ex vivo culture techniques to decipher signaling pathways involved in LG development.

The lacrimal gland (LG) secretes aqueous tears necessary for maintaining the structure and function of the cornea, a transparent tissue essential for ...

Cryosectioning Method for Microdissection of Murine Colonic Mucosa

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The colonic epithelium is dynamically turned over and epithelial cells are generated in the stem cell containing crypts of Lieberk&#252;hn. Progenitor cells produced in the crypt-bases migrate toward the luminal surface, undergoing a process of cellular differentiation before being shed into the gut lumen. In order to study these processes at the molecular level, we have developed a simple method for the microdissection of two spatially distinct regions of the colonic mucosa; the proliferative crypt zone, and the differentiated surface epithelial cells. Our objective is to isolate specific crypt and surface epithelial cell populations from mouse colonic mucosa for the isolation of RNA and protein.

Here we describe a simple method for the isolation of spatially distinct murine colonic epithelial surface cells from the underlying crypt-base cells.

The colonic mucosal tissue provides a vital barrier to luminal antigens. This barrier is composed of a monolayer of simple columnar epithelial cells. The ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.