我们感谢玉米 Borok 和晃平 Miyazono 为编写手稿提出建议。H.I.S. 和 M.H. 得到了上原纪念基金会研究金的支持, H.I.S. 得到了留学 Hayaishi 纪念奖学金的支持。这项工作得到了武田科学基金会 (犍为县) 的资助。

1. EndMT 的诱导
<ol><li>在标准培养条件下保持 MS-1 细胞, 避免融合。在材料表中描述了 MS-1 细胞的来源。对于 MS-1 细胞, 使用最低的基本中α (内存α) 与10% 胎小牛血清 (FCS), 50 毫升/ml 青霉素, 50 微克/毫升链霉素。</li>
	<li>用1x 磷酸盐缓冲盐水 (PBS) 在10厘米的盘子上洗 MS-1 细胞, 并在盘子中加入1.0 毫升的胰蛋白酶。孵育5分钟, 在37摄氏度。</li>
	<li>使用9毫升培养基分离细胞。收集细胞悬浮在15毫升管。</li>
	<li>在室温下离心 300–400 x g的细胞悬浮液5分钟。</li>
	<li>小心取出上清液, 在预热培养基中悬浮细胞颗粒。</li>
	<li>使用台盼蓝溶液和标准 hemocytometer 或自动单元计数器计数可行细胞。</li>
	<li>板 MS-1 细胞到未涂布的标准培养板上, 每 cm2 0.5 x 103细胞, 用于长期培养。例如, 板 5.0 x 103细胞每井在6个井文化板材。使用相同的文化媒体, 包括同一浓度的 FCS。在 MS-1 细胞中, TGF β诱导 EndMT 在长期培养 (至少 48–72 h) 与使用 FCS。</li>
	<li>刺激内皮细胞与 TGF-β2 (最终浓度为 1 ng/毫升) 后24小时。</li>
	<li>在一个湿润的孵化器中培养细胞 (5% CO2, 37 °c)。在关注形态学变化时, 每天监视细胞。</li>
	<li>在长期培养的 TGF 中, 用含有 TGF-β2的新鲜的预热培养基取代培养基, 并在48小时后进行治疗。</li>
	<li>进行下游分析。典型地, 在24小时治疗后观察肌动蛋白重组, 并观察 TGF β48–72治疗后下调内皮标记物和上调间充质标志物。</li>
</ol>2. 免疫细胞化学分析
<ol><li>在标准培养条件下保持 MS-1 细胞。</li>
	<li>板 MS-1 细胞到4井细胞培养室幻灯片在 1.0 x 103细胞每井 (1.7 厘米2)。幻灯片预先涂上明胶。每井都使用0.5–1.0 培养基。</li>
	<li>刺激内皮细胞与 TGF-β2 (最终浓度为 1 ng/毫升) 后24小时。当分析岩石在 EndMT 中的参与时, 添加一个岩石抑制剂 Y-27632 (10 微米) 1 小时之前, TGF β治疗。当评估 TGF β信号抑制的效果时, TGF β受体激酶抑制剂可以被使用。</li>
	<li>在一个湿润的孵化器中培养细胞 (5% CO2, 37 °c)。肌动蛋白重组是观察 MS-1 细胞后24小时的治疗与 TGF β。其他变化在48–72治疗后变得明显。对于 72 h 培养, 用 TGF β治疗后用含有 TGF β的新鲜培养基取代培养基。</li>
	<li>
f-肌动蛋白染色
	<ol>
<li>经过24小时的 TGF 治疗, 去除培养基, 固定细胞与4% 多聚甲醛 1x PBS 在室温下20分钟, 并冲洗细胞与 1x PBS。</li>
		<li>在室温下孵育0.2% 海卫 X-100 5 分钟。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>罗丹明-四甲基罗丹明 B 异硫氰酸酯与鹅蕈稀释150倍, 室温下阻塞缓冲1小时。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>室温下用菁核酸结合染料将细胞核染色5分钟。</li>
		<li>使用幻灯片安装介质冲洗幻灯片并在幻灯片上安装盖玻片面。</li>
		<li>用共焦显微镜观察。使用 543 nm 激光和560–615纳米发射过滤器检测罗丹明的荧光。使用 633 nm 激光和发射过滤器长于 650 nm 的核染色。经 TGF β治疗后, 可观察到厚应力纤维的形成。</li>
	</ol>
</li>
	<li>
VE 钙粘蛋白与α SMA 染色
	<ol>
<li>经过72小时的 TGF 治疗, 去除培养基, 固定细胞与冷50% 甲醇和50% 丙酮 (0.5–1.0 毫升每井) 为5分钟。</li>
		<li>冲洗细胞三倍 1x PBS (0.5–1.0 毫升每井)。</li>
		<li>根据制造商推荐的浓度, 在4摄氏度的黑暗中, 以主要抗体孵化, 在夜间阻塞缓冲区。使用 VE 钙黏蛋白单克隆抗体 (BV13, 1:100 稀释) 和 Cy3-conjugated α SMA 单克隆抗体 (1A4, 1:200 稀释)20。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>用绿色染料共轭二级抗体孵育细胞, 在1小时的室温下, 根据制造商推荐的浓度对 VE 粘附蛋白抗体进行阻塞缓冲。</li>
		<li>用 1x PBS 冲洗细胞三次。</li>
		<li>室温下用菁核酸结合染料将细胞核染色5分钟。</li>
		<li>使用幻灯片安装介质冲洗幻灯片并在幻灯片上安装盖玻片面。</li>
		<li>用共焦显微镜观察。使用 543 nm 激光和 560–615 nm 的发射过滤器检测α SMA (Cy3)。使用 488 nm 激光和505–550纳米的发射过滤器检测 VE 钙黏蛋白。通常情况下, TGF 治疗后, VE 钙黏蛋白信号的减少和α SMA 信号的增加将被观察到。</li>
	</ol>
</li>
</ol>3. 三维胶原凝胶收缩试验
<ol><li>在胶原凝胶收缩试验前, 用/不 TGF β MS-1 细胞培养72小时。</li>
	<li>在冰上准备 i 型胶原凝胶。混合冷胶原溶液, 10x 浓缩的记忆培养基, 胶原稀释缓冲液, 含 0.05 N 氢氧化钠, 2.2% NaHCO3, 200 毫米 HEPES pH 7.4 在 8:1: 1 比例。</li>
	<li>混合200毫升控制或 TGF β处理内皮细胞悬浮 (1.0 x 106 cells/200 ul) 和 800 ul 的胶原凝胶溶液。对于 TGF β处理的样品, 添加 TGF-β2 (1 ng/毫升) 的细胞悬浮。</li>
	<li>加入1.0 毫升的混合物, 每井12井培养板, 并允许固化在孵化器37°c 为30–60分钟。</li>
	<li>糊化后, 覆盖1.0 毫升的记忆α含有 10% FCS, 50 U/毫升青霉素, 50 微克/毫升链霉素, 以漂浮凝胶。对于 TGF β处理的样品, 添加 TGF-β2 (1 ng/毫升) 的媒体。 注: 对于 TGF β治疗样品, 添加 TGF β的凝胶和培养基。</li>
	<li>在37摄氏度的48小时内孵化漂浮凝胶。</li>
	<li>使用扫描仪扫描印版底部的凝胶图像。或者, 用数码相机在凝胶上方的固定距离上记录凝胶的图像。</li>
	<li>使用 ImageJ 对基于像素数的凝胶表面积进行量化。选择使用手绘选择或相关选择工具的凝胶, 或选择凝胶使用 "图像 |调整 |颜色阈值 "工具, 然后确定使用的凝胶面积" 分析 |度量 "命令。</li>
</ol>4. 锁定核酸基 miRNA 抑制剂对 miRNA 活性的抑制作用
<ol><li>在标准培养条件下保持 MS-1 细胞。</li>
	<li>染锁定核酸 (低噪声放大器) miRNA 抑制剂, 合成 miRNA 复式或 siRNAs (20 或 50 nM) 根据制造商的指示使用 lipofection 试剂。我们在以前的报告26、27中描述了低噪声 miRNA 抑制剂、合成 miRNA 复式和 siRNAs 的详细资料和来源。在 MS-1 细胞中, 标准的转染程序的基础上制造商的指导工作很好, 引入低噪声 miRNA 抑制剂, 合成 miRNA 复式或 siRNAs。</li>
	<li>后 16–48 h, 刺激内皮细胞与 TGF-β2 (最终浓度 1 ng/毫升)。</li>
	<li>进行各种下游分析, 包括 rna 的表达分析 (定量 rt-pcr 和 rna 序列分析), 免疫细胞化学分析, 和记者化验。我们在过去的报告20、26、27中描述了下游分析。</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial-mesenchymal+transition+of+brain+endothelial+cells:+possible+role+during+metastatic+extravasation.">Endothelial-mesenchymal transition of brain endothelial cells: possible role during metastatic extravasation.</a> PLoS One. 10 (3), e0119655 (2015).</li><li>Arciniegas, E., Sutton, A. B., Allen, T. D., Schor, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+growth+factor+beta+1+promotes+the+differentiation+of+endothelial+cells+into+smooth+muscle-like+cells+in+vitro.">Transforming growth factor beta 1 promotes the differentiation of endothelial cells into smooth muscle-like cells in vitro.</a> Journal of Cell Science. 103 (Pt 2), 521-529 (1992).</li><li>Deissler, H., Deissler, H., Lang, G. K., Lang, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGFbeta+induces+transdifferentiation+of+iBREC+to+alphaSMA-expressing+cells.">TGFbeta induces transdifferentiation of iBREC to alphaSMA-expressing cells.</a> International Journal of Molecular Medicine. 18 (4), 577-582 (2006).</li><li>Paranya, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aortic+valve+endothelial+cells+undergo+transforming+growth+factor-beta-mediated+and+non-transforming+growth+factor-beta-mediated+transdifferentiation+in+vitro.">Aortic valve endothelial cells undergo transforming growth factor-beta-mediated and non-transforming growth factor-beta-mediated transdifferentiation in vitro.</a> American Journal of Pathology. 159 (4), 1335-1343 (2001).</li><li>Maleszewska, 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=IL-1beta+and+TGFbeta2+synergistically+induce+endothelial+to+mesenchymal+transition+in+an+NFkappaB-dependent+manner.">IL-1beta and TGFbeta2 synergistically induce endothelial to mesenchymal transition in an NFkappaB-dependent manner.</a> Immunobiology. 218 (4), 443-454 (2013).</li><li>Ubil, 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=Mesenchymal-endothelial+transition+contributes+to+cardiac+neovascularization.">Mesenchymal-endothelial transition contributes to cardiac neovascularization.</a> Nature. 514 (7524), 585-590 (2014).</li><li>Xu, X., 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=Epigenetic+balance+of+aberrant+Rasal1+promoter+methylation+and+hydroxymethylation+regulates+cardiac+fibrosis.">Epigenetic balance of aberrant Rasal1 promoter methylation and hydroxymethylation regulates cardiac fibrosis.</a> Cardiovasc Research. 105 (3), 279-291 (2015).</li><li>Xiao, 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=Tumor+Endothelial+Cells+with+Distinct+Patterns+of+TGFbeta-Driven+Endothelial-to-Mesenchymal+Transition.">Tumor Endothelial Cells with Distinct Patterns of TGFbeta-Driven Endothelial-to-Mesenchymal Transition.</a> Cancer Research. 75 (7), 1244-1254 (2015).</li></ol>

作者没有什么可透露的。

据报道, 活化 Ras 和 TGF β治疗24小时诱导 EndMT 在 MS-1 细胞, 而 TGF β单独未能诱导 EndMT 在这短短的时间21。我们一贯地观察到, TGF β在 MS-1 细胞中的长时间治疗 (48–72 h) 后, 极大地诱发了 EndMT20。EndMT 在 TGF β (2–6天) 在各种内皮细胞如人脐静脉内皮细胞 (HUVEC)、人皮肤微血管内皮细胞 (HCMEC)、人冠状动脉内皮细胞等的长期治疗后反复观察 (HCEC), 小鼠肺内皮细胞 (《电...

内皮-间质转移 (EndMT) 是一种分化的内皮细胞经历多种分子变化的过程, 导致成纤维细胞样间质细胞1。EndMT 最初被描述作为内皮细胞变革在心脏的发展期间2,3。在早期心脏发育中, 心脏管由内心内膜和外心肌组成。这两层被一层细胞外基质所分离, 称为心脏果冻。胚胎心内膜细胞, 获得内皮细胞标志物, 转运到间充质细胞, 侵入底层的心脏果冻, 促进心脏垫的形成, 为房室瓣膜和隔膜提供基础和半月形阀门。此外, EndMT 已被认为是毛细血管和血管平滑肌细胞在其他胚胎血管系统的来源, 包括冠状动脉血管, 腹主动脉, 肺动脉4,5,6。此外, EndMT 与生理血管生成7有牵连。
积累的证据表明, EndMT 也参与多种心血管疾病和其他疾病1,8。EndMT 相关的条件包括血管钙化, 动脉粥样硬化, 肺动脉高压, 海绵状畸形, 器官纤维化, 静脉移植重塑, 移植肾功能障碍和癌症8, 9,10,11,12,13,14,15,16,17, 18. 最近的一份报告指出, 几种分子 EndMT 标记物可以作为诊断和预测肾脏移植肾功能障碍的一种工具,17。EndMT 相关的细胞信号通路的调制已被证明可以改善多种疾病的条件, 包括心脏纤维化和静脉移植重塑的动物模型8,15。因此, 了解 EndMT 所依据的机制对于制定针对 EndMT 的诊断和治疗策略是很重要的。
EndMT 的特点是细胞连接的丧失, 迁徙潜能的增加, 内皮特异基因的下调, 如 VE 钙粘蛋白, 上调间充质基因, 包括α平滑肌肌动蛋白 (α SMA)。此外, EndMT 和上皮间质转移 (急诊室), 一个类似的过程, 将上皮细胞转化为间充质细胞, 与各种细胞外基质成分的改变产生相关, 这可能有助于发展组织纤维化8,19。
最近, EndMT 的几项体外研究已经阐明了 EndMT15、20的分子机制的细节。EndMT 是由各种信号通路, 包括转化生长因子 (TGF) β, Wnt, 和缺口1诱导。其中, TGF β在急救和 EndMT 诱导中起着举足轻重的作用。在 EndMT, 长期暴露于 TGF β的结果 EndMT 在各种内皮细胞, 而短期暴露似乎不足21。我们这里描述了一个简单的 EndMT 诱导协议, 其中英里斯文 1 (MS-1) 小鼠胰腺微血管内皮细胞在长时间接触 TGF β20后,体外EndMT。在该模型中, 可以进行多种下游分析, 以研究 EndMT 的标志性特征, 包括形态学变化、内皮标记下调、间充质标志物上调和炎症基因骨架重排, 胶原凝胶收缩。
MicroRNAs (miRNAs) 是 22 nt 小规 rna, 直接转录后水平压制各种 mRNA 目标22,23。通过种子序列介导的目标识别, miRNAs 抑制数以百计的靶基因, 调节细胞分化、增殖和运动等多种细胞功能。这也是对急诊医师和 EndMT 的监管, 并且有几个 miRNAs 被报告为急诊医师和 EndMT24,25的监管者。本文提出的 EndMT 模型可以很容易地结合 miRNA 调制程序来测试 miRNAs 在 EndMT 中的作用。本文综述了 TGF β诱导 EndMT 在 MS-1 细胞中的实验方法, 并对其他内皮细胞中 TGF β的 EndMT 诱导条件进行了比较。

<table><tbody><tr><td>MS-1 cells</td><td>American Type Culture Collection</td><td>CRL-2279</td><td></td></tr><tr><td>MEM-alpha</td><td>Thermo Fisher Scientific</td><td>32571036</td><td></td></tr><tr><td>TGF-beta2</td><td>R&amp;amp;D</td><td>302-B2-002</td><td></td></tr><tr><td>4 well Lab-Tek II Chamber Slide</td><td>Thermo Fisher Scientific</td><td>154526</td><td></td></tr><tr><td>Y-27632&amp;nbsp;</td><td>Sigma-Aldrich</td><td>Y0503</td><td></td></tr><tr><td>Blocking One</td><td>nacalai tesque</td><td>03953-95</td><td></td></tr><tr><td>phalloidin-tetramethylrhodamine B isothiocyanate</td><td>Sigma-Aldrich</td><td>P1951</td><td></td></tr><tr><td>TOTO-3 iodide</td><td>Thermo Fisher Scientific</td><td>T3604</td><td></td></tr><tr><td>VE cadherin monoclonal antibody (BV13)</td><td>Thermo Fisher Scientific</td><td>14-1441-82</td><td></td></tr><tr><td>alpha-SMA Cy3 monoclonal antibody (1A4)</td><td>Sigma-Aldrich</td><td>C6198</td><td></td></tr><tr><td>Alexa Fluor 488 goat anti-mouse IgG (H+L)</td><td>Thermo Fisher Scientific</td><td>A-11001</td><td></td></tr><tr><td>Cover slip</td><td>Thermo Fisher Scientific</td><td>174934</td><td></td></tr><tr><td>Collagen solution</td><td>Nitta gelatin Inc.</td><td>Cellmatrix I-P</td><td></td></tr><tr><td>Collagen dilution buffer</td><td>Nitta gelatin Inc.</td><td>Cellmatrix I-P</td><td></td></tr><tr><td>LNA miRNA inhibitor</td><td>EXIQON&amp;nbsp;</td><td>miRCURY LNAmicroRNA Power Inhibitor (Negative Control B and target miRNA)</td><td></td></tr><tr><td>synthetic miRNA duplex</td><td>Qiagen&amp;nbsp;</td><td>miScript miRNA Mimic</td><td></td></tr><tr><td>Lipofectamine RNAiMAX</td><td>Thermo Fisher Scientific</td><td>13778030</td><td></td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668027</td><td></td></tr></tbody></table>

molecular analysis of endothelial-mesenchymal transition induced by transforming growth factor-&#946; signaling

内皮细胞表型可塑性是心血管系统发展、心血管疾病以及与器官纤维化相关的各种条件的基础。在这些条件下, 分化的内皮细胞获得间充质样表型。这一过程称为内皮-间质转移 (EndMT), 其特点是下调的内皮标记, 上调的间充质标记, 和形态学的变化。EndMT 是由几种信号通路引起的, 包括转化生长因子 (TGF) β、Wnt 和凹槽, 并由类似于上皮-间质转移 (急诊) 的分子机制所调控, 对肠、组织纤维化和肿瘤转移。了解 EndMT 的机制对于发展针对 EndMT 的诊断和治疗方法非常重要。EndMT在体外的鲁棒诱导是一种常见的基因表达特征, 识别 druggable 分子机制和 EndMT 调节剂的筛查。在这里, 我们描述了一种诱导 EndMT 的体外方法。MS-1 小鼠胰腺微血管内皮细胞在长时间暴露于 TGF β后 EndMT, 显示上调的间充质标志物和形态学变化以及诱导多种炎症趋化因子和细胞因子。microRNA (miRNA) 调制的分析方法也包括在内。这些方法为调查 EndMT 的机制和 miRNAs 对 EndMT 的贡献提供了一个平台。

TGF β是 EndMT 在各种内皮细胞中的有力诱导剂。在对 MS-1 细胞进行24小时的 TGF 治疗后, 对 F 肌动蛋白的染色显示重组肌动蛋白应激纤维 (图 1A)20。用岩石抑制剂 Y-27632 预处理抑制肌动蛋白重组20的诱导。MS-1 内皮细胞从经典的鹅卵石状形态学转变为 TGF β治疗时的间充质纺锤形形态 (图 1B)。TGF β?...

Watch this Scientific Journal Video about Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-Î² Signaling at JoVE.com

Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-&#946; Signaling

本文介绍了一种体外诱导内皮-间质转移 (EndMT) 的方法, 该协议对于研究 EndMT 中涉及的细胞信号通路是有用的。在该实验模型中, EndMT 是由 TGF β在 MS-1 内皮细胞中的治疗引起的。

转化生长因子β信号诱导内皮-间质转移的分子分析

Phenotypic plasticity of endothelial cells underlies cardiovascular system development, cardiovascular diseases, and various conditions associated with ...

molecular-analysis-endothelial-mesenchymal-transition-induced

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K 次观看.  Massachusetts Institute of Technology. 本文介绍了一种体外诱导内皮-间质转移 (EndMT) 的方法, 该协议对于研究 EndMT 中涉及的细胞信号通路是有用的。在该实验模型中, EndMT 是由 TGF β在 MS-1 内皮细胞中的治疗引起的。

视频: Molecular Analysis of Endothelial-mesenchymal Transition Induced by Transforming Growth Factor-β Signaling

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

科研

发育生物学

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.

在肉瘤细胞间质上皮转变的诱导

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

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.

研究TGF-β信号和TGF-β引起的乳腺癌和正常细胞的上皮到中位过渡

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

癌症研究

Visualization and Quantification of TGF&#946;/BMP/SMAD Signaling under Different Fluid Shear Stress Conditions using Proximity-Ligation-Assay

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and homeostasis of the vasculature system Therefore, deregulation in this signaling pathway results in severe vascular pathologies, such as pulmonary artery hypertension, hereditary hemorrhagic telangiectasia, and atherosclerosis. Endothelial cells (ECs), as the innermost layer of blood vessels, are constantly exposed to fluid shear stress (SS). Abnormal patterns of fluid SS have been shown to enhance TGF&#946;/BMP signaling, which, together with other stimuli, induce atherogenesis. In relation to this, atheroprone, low laminar SS was found to enhance TGF&#946;/BMP signaling while atheroprotective, high laminar SS, diminishes this signaling. To efficiently analyze the activation of these pathways, we designed a workflow to investigate the formation of transcription factor complexes under low laminar SS and high laminar SS conditions using a commercially available pneumatic pump system and proximity ligation assay (PLA).
Active TGF&#946;/BMP-signaling requires the formation of trimeric SMAD complexes consisting of two regulatory SMADs (R-SMAD); SMAD2/3 and SMAD1/5/8 for TGF&#946; and BMP signaling, respectively) with a common mediator SMAD (co-SMAD; SMAD4). Using PLA targeting different subunits of the trimeric SMAD-complex, i.e., either R-SMAD/co-SMAD or R-SMAD/R-SMAD, the formation of active SMAD transcription factor complexes can be measured quantitatively and spatially using fluorescence microscopy.
The usage of flow slides with 6 small parallel channels, that can be connected in series, allows for the investigation of the transcription factor complex formation and inclusion of necessary controls. 
The workflow explained here can be easily adapted for studies targeting the proximity of SMADs to other transcription factors or to transcription factor complexes other than SMADs, in different fluid SS conditions. The workflow presented here shows a quick and effective way to study the fluid SS induced TGF&#946;/BMP signaling in ECs, both quantitatively and spatially.

Here, we establish a protocol to simultaneously visualize and analyze multiple SMAD complexes using proximity ligation assay (PLA) in endothelial cells exposed to pathological and physiological fluid shear stress conditions.

使用接近-连接-测定法对不同流体剪切应力条件下TGFβ/BMP/SMAD信号传导的可视化和定量

Transforming Growth Factor &#946; (TGF&#946;)/Bone Morphogenetic Protein (BMP) signaling is tightly regulated and balanced during the development and ...

生物化学

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

医学

High Throughput Traction Force Microscopy Using PDMS Reveals Dose-Dependent Effects of Transforming Growth Factor-&#946; on the Epithelial-to-Mesenchymal Transition

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and mechanical stability, and represents a core element of multi-cellular animal life. In adherent cells, acto-myosin contraction is seen in traction forces that cells exert on their substrate. Dysregulation of cellular contractility appears in a myriad of pathologies, making contractility a promising target in diverse diagnostic approaches using biophysics as a metric. Moreover, novel therapeutic strategies can be based on correcting the apparent malfunction of cell contractility. These applications,&#160;however, require direct quantification of these forces.
We have developed silicone elastomer-based traction force microscopy (TFM) in a parallelized multi-well format. Our use of a silicone rubber, specifically polydimethylsiloxane (PDMS), rather than the commonly employed hydrogel polyacrylamide (PAA) enables us to make robust and inert substrates with indefinite shelf-lives requiring no specialized storage conditions. Unlike pillar-PDMS based approaches that have a modulus in the GPa range, the PDMS used here is very compliant, ranging from approximately 0.4 kPa to 100 kPa. We create a high-throughput platform for TFM by partitioning these large monolithic substrates spatially into biochemically independent wells, creating a multi-well platform for traction force screening that is compatible with existing multi-well systems.
In this manuscript, we use this multi-well traction force system to examine the Epithelial to Mesenchymal Transition (EMT); we induce EMT in NMuMG cells by exposing them to TGF-&#946;, and to quantify the biophysical changes during EMT. We measure the contractility as a function of concentration and duration of TGF-&#946; exposure. Our findings here demonstrate the utility of parallelized TFM in the context of disease biophysics.

We present a high throughput traction force assay fabricated with silicone rubber (PDMS). This novel assay is suitable for studying physical changes in cell contractility during various biological and biomedical processes and diseases. We demonstrate this method's utility by measuring a TGF-&#946; dependent increase in contractility during the epithelial-to-mesenchymal transition.

使用 PDMS 的高通量牵引力显微镜揭示了转化生长因子的剂量依赖效应 - 对中皮到中位过渡的影响

Cellular contractility is essential in diverse aspects of biology, driving processes that range from motility and division, to tissue contraction and ...

生物工程

Strategic Endothelial Cell Tube Formation Assay: Comparing Extracellular Matrix and Growth Factor Reduced Extracellular Matrix

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by hijacking the process of angiogenesis, in which new blood vessels are formed from existing blood vessels. The CXCR2 (chemokine (C-X-C motif) receptor 2) receptor is a transmembrane G-protein-linked molecule found in many cells that is closely associated with angiogenesis1. Specific blockade of the CXCR2 receptor inhibits angiogenesis, as measured by several assays such as the endothelial tube formation assay. The tube formation assay is useful for studying angiogenesis because it is an excellent method of studying the effects that any given compound or environmental condition may have on angiogenesis. It is a simple and quick in vitro assay that generates quantifiable data and requires relatively few components. Unlike in vivo assays, it does not require animals and can be carried out in less than two days. This protocol describes a variation of the extracellular matrix supporting endothelial tube formation assay, which tests the CXCR2 receptor.

This manuscript describes a tube formation assay to quantify the effects of a given compound or condition on angiogenesis by using endothelial cell tube formation in a controlled environment.

战略内皮细胞形成实验:比较外基质和生长因子，细胞外基质

Malignant tumors require a blood supply in order to survive and spread. These tumors obtain their needed blood from the patient's blood stream by ...

Analyzing Angiogenesis: In Vitro Assay for Tube Formation on BM Matrix

Trophoblast Cell Recovery from Angiogenesis-Tube Formation Assay for Differentiation Marker Expression Analysis

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of mesenchymal to endothelial-like transition. Traditionally, this process is evaluated by an in vitro tube-formation assay, where the cells organize themselves into tube-like structures when seeded over a polymerized basement membrane preparation. Although several structural features can be measured in photomicrographs of the structures, to assess the real commitment of EVT to the endothelial-type phenotype, biochemical analysis of cell extracts is required. Scraping the cells from the culture dish to obtain RNA and/or protein extracts is not an alternative since the tube-like structures are severely contaminated by the bulk of proteins from the polymerized basement membrane. Thus, a strategy to separate the cells from the basement membrane proteins prior to the preparation of cell extracts is needed. Here, a simple, fast, and cost-effective method to recover the tube-like structures from the in vitro tube-formation assay and the subsequent analysis by biochemical techniques is presented. Tube-like structures formed by HTR8/SVneo cells, an EVT cell line, were liberated from the polymerized basement membrane by a short incubation with PBS supplemented with ethylenediaminetetraacetic acid (EDTA). After serial washes, a ready-to-use pellet of purified tube-like structures can be obtained. This pellet can be subsequently processed to obtain RNA and protein extracts. qPCR analysis evidenced the induced expression of VE-cadherin and alphav-integrin, two endothelial cell markers, in EVT-derived tube-like structures compared to control cells, which was consistent with the induction of the endothelial cell marker, CD31, evaluated by immunofluorescence. Western blot analysis of the tube-like structures' protein extracts revealed the overexpression of RECK in transfected HTR8/SVneo cells. Thus, this simple method allows to obtain cell extracts from the in vitro tube-formation assay for the subsequent analysis of RNAs and protein expression.

The present protocol allows the recovery of tube-like structures formed in the classical in vitro angiogenesis-tube formation assay where cells, such as trophoblast cells, organize in capillary-like structures over a layer of extracellular basement membrane matrix. The recovered structures are used for RNA and protein isolation for various biochemical analyses.

During placenta development, extravillous trophoblast (EVT) cells invade the maternal decidua to remodel the uterine spiral arteries by a process of ...

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.

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 ...

生物学

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.