作者要感谢朱莉娅Hatler和马丁拉姆斯登（R＆D系统）的有益讨论和审稿。

1。诱导的EMT <ol><li>温暖的培养基至37℃的水浴中。所用的培养基是相同的用于您所关注的细胞的标准培养介质。例如，A549人肺癌细胞系生长在Kaighn氏F12补充10％胎牛血清和2mM谷氨酰胺。 </li><li>利息用解离液（ 如用TrypLE快递）收获细胞。 </li><li>悬浮在15毫升锥形管中的细胞在预热的培养基。 </li><li>离心细胞悬浮液以400×g离心5分钟。 </li><li>小心地倒入垃圾容器中取出上清液。 </li><li>悬浮在预热的培养基将细胞沉淀。 </li><li>用台盼蓝计数活细胞。除去细胞悬浮液的样品和稀释在0.4％锥虫蓝溶液。将10微升稀释样品的血球计数及活细胞（细胞不变成蓝色）。用细胞计数来确定小区D密度的解决方案。 </li><li>板细胞到组织培养物处理板或培养瓶在每平方厘米0.9〜1.0×10 4个细胞。例如，0.5×10 6细胞接种于100毫米的钢板在含1X EMT诱导培养基添加剂（6毫升媒体每100mm板）文化传媒。 </li><li>培养接种的细胞在37℃/ 5％CO 2的培养箱中培养。每天监测细胞形态​​。 </li><li>电镀后三天，取出纸张，换用含1X EMT诱导培养基补充新鲜预热的培养基。继续培养在37℃/ 5％CO 2培养箱。 </li><li>电镀后五天，细胞准备用于分析。细胞形态由倒置显微镜观察。 </li></ol> 2。通过免疫细胞蛋白表达的分析<ol><li>通过放置盖玻片在含95％乙醇的培养皿制备无菌12毫米盖玻片的24孔板中。轻轻地从我们的乙醇去除盖玻片ing一针和弯钳和火焰灭菌。放置无菌盖玻片到24孔板的一个孔中。重复剩余盖玻片。 </li><li>准备细胞悬液中1.1节描述 - 1.7。 </li><li>板1.6×10 4个细胞/孔在0.5ml含1X EMT诱导培养基补充预热的培养基。 </li><li>增长和在部分1.9中描述养活细胞 - 1.11。 </li><li>电镀后五天，除去介质和修复的细胞用300μl/孔的4％多聚甲醛在1X磷酸盐缓冲盐水（PBS）中20分钟，在室温下进行。 </li><li>除去固定剂和冲洗细胞2倍用1X PBS，500μL/孔。 </li><li>培养细胞中加入400μl阻断缓冲液/孔（1X PBS中含有1％牛血清白蛋白（BSA），10％正常驴血清和0.3％的Triton X-100）1小时，在室温下进行。 </li><li>在制造商中孵育一抗在400微升/推荐浓度以及封闭液3小时，在室温温度场的e或过夜，4°C。当使用直接缀合到荧光染料的初级抗体，孵育在黑暗中的样品。 </li><li>在500μl的1×PBS /孔的含有0.1％BSA，每次5分钟洗涤液洗涤细胞3次。当使用直接进行荧光标记一抗，直接进行步骤2.12。 </li><li>在400μl的1×PBS /孔的含有1％BSA进行1小时，在室温下在黑暗中孵育细胞中二级抗体在制造商推荐的浓度</li><li>在500μl的1×PBS /孔的含有0.1％BSA，每次5分钟洗涤液洗涤细胞3次。 </li><li>如果需要的话，对细胞核用DAPI溶液。 </li><li>冲洗细胞，去离子水和盖玻片安装面朝下使用安装媒体的幻灯片。 </li></ol>

<ol>
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以前的方法诱导的EMT一般都用TGF-β刺激或遗传修饰。虽然单独的TGF-β已被证明是诱导EMT在一些细胞类型中，现已证实，TGF-β是必要的EMT的，但它不足以在所有细胞类型6,14。使用遗传修饰为EMT诱导是耗时且劳动密集型的。这里描述的方法使用的因素，包括抗人E-钙粘蛋白，抗人SFRP-1，抗 - 人DKK-1，重组人的Wnt-5a和重组人TGF-β1能可靠地诱导EMT的组合。这些因素的组合的目的是阻止的E-钙粘蛋白为基础的粘合性，为EMT诱导4的一个关键步骤，并通过添加的Wnt-5a中的蛋白质，以及使用阻断抗体对Wnt信号的抑制剂增强Wnt信号传导，SFRP-1和DKK -1。 Wnt信号和TGF-β信号已被证明在自分泌环采取行动，以诱导和维持的间充质细胞统计E 6。这个感应方法避免了遗传操作，并且比使用TGF-β的单独更有效。重要的是，我们能够观察到诱导EMT的细胞类型，包括MCF7和PANC-1细胞，先前已经证明不是完全响应的TGF-β刺激（ 图5）14。 细胞的EMT诱导培养基补充节目少紧凑和增加纺锤状的形态（ 图1）处理。此外，还有减少表面的E-钙粘蛋白表达的并发与增加纤维连接蛋白的表达，这是EMT（ 图2和3）的标志。额外的间充质标记物如波形蛋白和蜗牛也上调了EMT诱导细胞相比，未诱导的对照（ 图4）。越来越多的迁移和侵袭能力的间充质细胞的主要功能特性。在体外 </em&gt;迁移和侵袭测定法，与EMT诱导培养基补充处理的细胞表现出的迁移和侵入（ 图6）显著增加容量。 如使用图7中所示的抗体阵列表达数据表明了这一点为EMT诱导简单方法是EMT的信号传导的研究是有用的。不同类型的细胞可以比前和后的EMT诱导获得与EMT相关的共同表达特征，例如和ERK1 / 2可以看出在两个MCF7和A549的EMT诱导的细胞增加的磷酸化CREB的。 CREB的S133的磷酸化刺激DNA结合并已经显示出作用，TGF-β1-诱导的EMT 15的下游。 ERK1 / 2的磷酸化也被证明充当TGF-β1-诱导的EMT 16的下游。在信号通路中的细胞类型的不同，也可阐明看到，在A549细胞与p70S6增加GSK-3β的磷酸化ķ磷酸化在MCF7。 GSK-3β磷酸化的丝氨酸9减小它的功能，和GSK-3β激酶已被证明能够抑制亲EMT的转录因子，蜗牛17。与此相反，p70S6K的诱导蜗牛的活性和它的磷酸化与活化该蛋白18的相关联。 总体而言，简单，可靠的诱导EMT的，特别是在先前表明不回应只对TGF-β细胞，是理解EMT的生物，确定共同的表达特征用作预后指标，以及开发和评估新的治疗癌症有用和纤维化疾病。

上皮间质转化（EMT）是由哪一个极化上皮细胞经历了各种产生了高度能动，成纤维细胞样细胞的间充质转变的过程。这个过程发生时，部分地通过基因表达的变化和粘着连接的降解，导致迁移和侵入的能力增强。生理上，EMT的胚胎发育和伤口愈合过程中起着重要的作用。适当控制EMT的损失可能导致纤维化和1,2癌转移。 E-钙粘蛋白的上皮细胞表面上的还原是在一个细胞通过EMT 3,4进展的关键一步。 E-钙粘蛋白是一种单次跨膜蛋白，其直接与邻近的细胞钙粘蛋白相互作用。除了其在细胞粘附作用，E-cadherin的影响，通过细胞的E-cadherin蛋白的胞质尾之间的信令交互哒各种调控蛋白，特别是β-连环蛋白。 β-catenin在粘着连接的稳定起到了重要作用。在经典Wnt信号，β-连环蛋白是从E-cadherin蛋白释放并转移到它充当Wnt信号通路3,4,5下游转录因子的细胞核。在细胞核中，β-连环蛋白已被证明能增加几个EMT相关的因素，包括扭，团状，纤连蛋白，以及多种基质金属蛋白酶3,6的转录。 除了​​Wnts，TGF-β信号已被证明是既在正常发育和疾病状态7,8,9中的EMT诱导中发挥显著作用。 TGF-β参与过程中腭的形成和发育的心脏10时所发生的EMT。它也被牵连纤维化和癌症进展到转移7。 这里所描述的公关程序ovides一致诱导EMT的多种细胞类型中，而不需要的遗传操作。这个程序使用的是E-钙粘蛋白的鸡尾酒，SFRP-1和DKK-1阻断抗体和Wnt-5a和TGF-β1的重组蛋白。这种鸡尾酒的目的是阻止E-cadherin蛋白为基础的附着力，同时增强的Wnt和TGF-β信号。的能力为健壮的EMT诱导对于理解这个过程的生物学和如何操纵它用于治疗目的的关键。 EMT的电流共同标记，E-钙粘蛋白（在EMT下调）和波形蛋白（上调EMT），可以发现共表达于癌症，因此不提供潜在的转移性细胞12的最佳的诊断标志物。重要的是，各种已经历EMT的细胞类型的分析是有用的，以开发可用于癌症和纤维化11诊断目的共同的基因表达特征。此外，最近的研究表明，电子MT可以在癌症干细胞的形成中发挥作用，这表明已经历EMT的细胞可以用于药物筛选可用于鉴定，可以针对这些细胞13的化合物。

<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

上皮间质转化（EMT）是在开发过程中适当的形态必不可少的。这个过程的错误调节已被认为在纤维化的关键事件和癌的进展为转移性状态。了解背后的EMT过程是刻不容缓的早期诊断和临床控制这些疾病状态。可靠的诱导EMT 的体外是药物发现的有用工具，以及确定共同的基因表达特征进行诊断。在这里，我们展示的诱导EMT中的各种细胞类型的一个简单方法。也包括方法细胞前和后的EMT诱导免疫细胞化学分析。此外，我们证明，通过基于抗体阵列分析和迁移/侵袭测定法本方法的有效性。

，此处所描述的EMT诱导培养条件下提供一种可靠的方法用于诱导EMT的多种细胞类型中的图1和图2展示的形态学和标记物表达水平为4种不同的人细胞系:T98G胶质瘤细胞，HT29结肠腺癌细胞，A549肺癌细胞，并MCF10A乳腺上皮细胞。这是与EMT的诱导培养基添加剂处理过的细胞从传统的上皮形态（ 图1A - 1D）变更为间质，纺锤状形态（ 图1E - 1H）。 EMT诱导的细胞似乎不太密集的排布紧密的殖民地相比，未诱导的细胞。未诱导MCF10A样本含有由多个松散的细胞包围排列紧密的集群。这些紧凑团簇的E-cadherin蛋白阳性（ 图2D），消失在治疗EMT的诱导培养基增刊键相（ 图2H）。 EMT还评估了上皮标记和间质标记物的表达上调下调。 E-cadherin表达的下调通常观察到在不同的细胞类型3,4以下的EMT诱导图2展示了E-cadherin蛋白的表面表达在大部分未经处理的细胞（ 图2A - 2D，红色）。相比，它的缺席后EMT的诱导（ 图2E - 2H;红色）。一个细胞系，T98G，发现有E-cadherin的前EMT的诱导，这排除了其分析的一个非常低的基础水平。间充质标志物，纤维连接蛋白的上调，也可见诱导EMT的诱导培养基添加剂（ 图2A - 2D与图2E - 2H;绿色）之后。 E-钙粘蛋白和纤维连接蛋白的表达水平见过用免疫细胞化学通过总细胞裂解物的蛋白质印迹（ 图3）被进一步证实。免疫印迹证实E-cadherin蛋白和纤维连接蛋白的表达看到的免疫细胞化学的上调下调。该乐队是用密度和标准化为GAPDH，以确定相对倍数变化下的EMT诱导定量。 ，E-cadherin蛋白水平分别降低6.4和3.4倍，在A549和MCF10A细胞分别。纤连蛋白水平增加4.6，4.1和2.3倍，T98G，A549，和MCF10A细胞。虽然印迹不显示在HT29细胞后诱导EMT显著减少E-钙粘蛋白的水平，该免疫细胞化学结果表明在E-钙粘蛋白（ 图2B与2F），这是无法在加以区别的表面表达减少总细胞裂解物的免疫印迹。 为了进一步评估EMT的状态，其他已知的标记S代表EMT分析前和后的EMT诱导。与以前的结果，E-cadherin的表达下调在检查了所有的细胞系，这表明EMT的诱导。此外，间充质标志物，波形蛋白和蜗牛，以下的处理与EMT诱导培养基补充剂（ 图4）被上调在A549，T98G，并MCF10A细胞。在HT29细胞系中没有显示出的这些间质标记物具有或不具有EMT诱导表达（数据未显示），这可能表明，这些细胞利用为EMT诱导替代途径。 尽管TGF-β是足以诱导EMT的某些细胞上下文7,8,9，其它细胞类型不完全去除了TGF-β14的响应。 MCF7人乳腺癌细胞和胰腺癌细胞系PANC-1人胰腺癌细胞中都报道不向TGF-β的单独信令14作出响应。要确定是否这两条线可能在VITR经历EMTO，细胞培养的EMT诱导培养基添加剂的存在。细胞用重组单独TGF-β1，在EMT诱导培养基补充内的浓度。这些细胞保留了上皮形态，组成细胞的紧密包装的菌落（数据未示出）和表面的E-钙粘蛋白水平相似，看到在对照细胞（ 图5C和图5D与图5A和5B）。与此相反，细胞EMT的诱导培养基补充刺激呈现在表面E-cadherin蛋白水平的显着降低（ 图5E和5F）。这些细胞也得到更多的间充质的形态，如细胞变少紧凑和更纺锤状（数据未显示）。这些结果表明，这里所描述的EMT诱导方法能够促进EMT的形态，通常以TGF-β-I抗性标记物的表达变化在细胞EP3受体激动剂诱导的EMT。 除了改变标志物表达，间充质细胞的另一特点是他们的迁移和侵袭能力。迁移和侵袭的细胞具有或不具有EMT诱导培养基补充，根据制造商的说明使用96孔BME细胞侵袭测定，分析培养的。简言之，将细胞接种到96孔板（50,000细胞/孔）含有过滤器滤芯具有8.0微米的孔隙。 48小时后，迁移是由钙黄绿素在曾透过过滤器迁移细胞的AM染色评估。将每个样品一式三份，结果相似测试两次。从一个实验的代表性结果示于图6A中 。统计学显著增加（P值&lt;0.003），在细胞迁移被认为与以下EMT诱导既A549和PANC-1细胞。为了评估这些细胞的侵入（ 即迁移至细胞外基质）的能力，在相同的测定是与基底膜提取物涂布的过滤器进行。 EMT诱导的细胞表现出统计学显著性（P值&lt;0.001）增加侵袭能力相比，未经处理的细胞， 如图6B所示。 鲁棒诱导EMT的是用于基因表达的改变和信令发生在这些细胞中的分析是有用的。使用来自两个处理过的和未处理的细胞裂解物的基于抗体阵列分析是一种方法来分析单个样品的各种分子的表达水平。作为一个例子，我们分析的磷酸化MAPK家族成员的水平裂解物从非诱导的和EMT诱导的MCF7和A549细胞用蛋白质组探查人类磷酸化MAPK阵列试剂盒。该数组是从具有相似结果的独立的EMT诱导处理执行两次。 图7示出了从一个阵列的代表性数据。这两种类型的细胞表现出CREB（S133）的磷酸化增加，ERK1（T2<html> 02/Y204）和ERK2（T185/Y187）在EMT诱导的细胞与对照组相比。在信号转导事件的差异两种细胞类型之间进行观察，以及。 A549细胞显示增加GSK-3β（S9）的磷酸化，而MCF7细胞呈增加的p70S6K（T421/S424）的磷酸化。 <img src="/files/ftp_upload/50478/50478fig1.jpg" alt="图1" fo:content-width="6in" fo:src="/files/ftp_upload/50478/50478fig1highres.jpg" fo:width="6in"/> 图1。刺激EMT的诱导培养基补充后的间充质形态感应（A - D）上皮的四个类型的细胞形态显示，连续5天在标准培养基不EMT的诱导培养基补充以下文化（E - H）。间充质的形态四种细胞类型的表示与EMT的诱导培养基补充培养5天。 NT"FO:保持together.within页="总是"&gt; <img src="/files/ftp_upload/50478/50478fig2.jpg" alt="图2" fo:content-width="6in" fo:src="/files/ftp_upload/50478/50478fig2highres.jpg" fo:width="6in"/> 图2。上皮细胞标志物的表达，并在EMT诱导骨髓间充质细胞标志物表达的上调下调。细胞免疫荧光染色检测上皮标志物E-钙粘蛋白（红色）的表达，以及间质标记物，纤维连接蛋白（绿色）（A - D）控制细胞无EMT的诱导培养基补充培养5天（E - H）细胞的EMT的诱导培养基补充培养5天。所有细胞经DAPI染色（蓝色）。 <img src="/files/ftp_upload/50478/50478fig3.jpg" alt="图3"/> 图3。用免疫印迹的上皮细胞和间充质标志物表达的确认<html> 。四个类型的细胞的总细胞裂解物的alysis Western印迹表明，与（EMT）或不处理（ - ）的EMT诱导培养基补充5天。频段的E-钙粘蛋白，纤连蛋白和GAPDH的装载控制是如左图所示。的大小标记物是，如右图所示。 <img src="/files/ftp_upload/50478/50478fig4.jpg" alt="图4"/> 图4。的间充质标志，波形蛋白和蜗牛在EMT诱导细胞上调。细胞的多色免疫荧光染色检测上皮标志物E-钙粘蛋白（灰色）的表达，以及间质标记，波形蛋白（绿色）和蜗牛（红） （A - C）控制细胞无EMT的诱导培养基补充剂培养5天。（D - F）细胞的EMT的诱导培养基补充培养5天。 lways"&gt; <img src="/files/ftp_upload/50478/50478fig5.jpg" alt="图5" fo:content-width="4in" fo:src="/files/ftp_upload/50478/50478fig5highres.jpg" fo:width="6in"/> 图5。诱导EMT的TGF-β1无响应的细胞。（A和B）的细胞与单媒体培养5天。（C和D）细胞在含TGF-β1的10毫微克/毫升，连续5天培养基。（E和F）中的EMT诱导培养基补充5天培养的细胞。所有的细胞被染色的E-cadherin的表达（红色）和经DAPI染色（蓝色）。 <img src="/files/ftp_upload/50478/50478fig6.jpg" alt="图6"/> 图6。 EMT诱导的细胞增加了迁移和侵袭能力。细胞培养后通过8微米孔径的过滤器，迁移48小时的A.平均数。的B.平均人数细胞能够通过侵入培养48小时后涂基底膜提取物8微米孔径的过滤器。误差棒表示超过3个孔的标准偏差。 <img src="/files/ftp_upload/50478/50478fig7.jpg" alt="图7"/> EMT诱导的图7。基于抗体的阵列表达分析。从A549（A和C）裂解液和MCF7（B和D）细胞有或无的EMT诱导培养基培养补充被用于利用蛋白质组探查人类磷酸激酶试剂盒阵列阵列分析。 的景点，突出和 B，然后使用密度定量。未诱导EMT诱导裂解的比较显示在下面（C和D）中相应的柱状图。 <a href="https://www.jove.com/files/ftp_upload/50478/50478fig7large.jpg" target="_blank">点击这里查看大图&lt;</a>/ A&gt;。

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

一个直接的方法被描述为诱导上皮至间质转变（EMT）在各种细胞类型。方法对细胞的EMT状态的免疫细胞化学分析也包括在内。

诱导上皮和分析间质转化

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

Research

JoVE Journal

Biology

35.6K 次观看.  R&D Systems, Inc.. 一个直接的方法被描述为诱导上皮至间质转变（EMT）在各种细胞类型。方法对细胞的EMT状态的免疫细胞化学分析也包括在内。 

视频: 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.

光/暗转换测试小鼠

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, ...

科研

生物学

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.

一个神经元和星形胶质细胞共培养检测神经毒性的高含量分析

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.

活细胞成像和胚胎上皮细胞的定量分析非洲爪蟾</em

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.

RNA纯化的染色质隔离（CHIRP）

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.

在毛发再生的上皮 - 间充质信号的快速遗传分析

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.

从成年小鼠切牙牙源性上皮干细胞的分离和培养

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.

检测选择性剪接的过程中上皮 - 间质转化

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.

操纵小鼠泪腺

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.

Cryosectioning方法小鼠结肠黏膜显微切割

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.