JAS确认来自杜克大学癌症研究所，杜克大学的泌尿生殖系肿瘤学实验室，骨科的杜克大学部的支持。 HL是由美国国家科学基金会（NSF）中心理论生物物理的支持（NSF PHY-1427654）和NSF DMS-1361411，并作为CPRIT（癌症预防和得克萨斯州的研究所）学者在得克萨斯州癌症研究在莱斯大学。 KEW由NIH F32 CA192630 MKJ和HL从与玛丽·C·法拉克-卡森，JN Onuchic，萨米尔·M·汉什，肯内特·J·皮塔，和唐纳德·S·科费有益的讨论中受益的支持。

1.试剂的配制<ol><li>通过添加胎牛血清（FBS）和5毫升青霉素 - 链霉素（5000 U / mL）添加到500mL的DMEM中的50毫升制备DMEM用于细胞培养。此介质可以被储存在4℃下进行长达六个月。 </li><li>重悬无核酸酶水冻干引物以10μM的终浓度。在-20℃储存再悬​​浮的引物。 </li><li>制备放射免疫沉淀测定（RIPA）缓冲液（150mM NaCl的，​​0.5％脱氧胆酸钠，0.1％SDS，50毫摩尔Tris，pH8.0）中。有蛋白酶抑制剂混合物补充使用前保持在冰上缓冲在使用时。储存于4℃。 </li><li>在水和注射器过滤器制备的10mg / mL凝聚胺（海美溴铵）用0.45μm聚醚砜过滤器进行灭菌。溶液可以储存在4℃下长达六个月。 </li><li>通过在10mL PBS中溶解10毫克制备的硝基蓝四唑氯化物和1mg / mL的工作溶液。储存于4℃。</li></ol> 2. GRHL2的慢病毒转导 1天 <ol><li>板每一个6孔板的孔3×10 5个HEK293T细胞于2ml补充的DMEM。量化使用自动细胞计数器细胞。过夜培养后60％汇合，在37℃，5％CO 2 -细胞应为40。 </li></ol> 第2天 <ol start="2"><li>稀释的2μg空载体的（的pCMV-UBC-EGFP）或2的pCMV-GRHL2-UBC-EGFP 24微克，25在无血清培养基中的200μL的1.8微克pΔ8.9和0.2微克的pCMV-VSV的沿-G辅助质粒。在一个单独的管中，在稀释每次转染无血清培养基（8μL在400μL对于两个样品）的200μL基于脂质的转染试剂的4μL。添加转染试剂混合物的200μL到每个质粒溶液，孵育在室温下20分钟。 </li><li>在incuba和灰，通过真空抽吸除去培养基，并用1毫升的PBS漂洗细胞洗HEK293T细胞。与无血清培养基的800微升更换PBS。在37℃下在5％CO 2将来自步骤2.2滴加质粒混合物到每个孔中，孵育细胞2小时:20分钟后，加入200μL转染试剂。 </li><li>小心地真空抽吸除去转染培养基，并用2毫升补充的DMEM替换。返回细胞的培养箱中进行过夜培养。 注:HEK293T细胞松散粘连。媒体更换必须谨慎进行以从盘或烧瓶的底部限制除去细胞。 </li></ol> 第3天 <ol start="5"><li>刷新转染的HEK293T细胞和平板培养基每一个6孔板的孔3×10 5 RD细胞在2mL补充的DMEM。培养细胞过夜。 RD细胞是市售的人横纹肌肉瘤细胞系。 </li></ol> 第4天 </p&gt; <ol start="6"><li>从HEK293T细胞收集病毒介质。移液器从HEK293T细胞和地点的DMEM培养基到一个新的15毫升锥形。新的DMEM媒体和地方HEK293T细胞放回孵化器为第二天小心地更换。 </li><li>添加10毫克/每毫升ml聚凝胺病毒介质的2μL成两个新的50个毫升锥形管中（一个用于空载体转染和另一个用于GRHL2转染）。注射器过滤病毒介质通过从3mL注射器在柱塞和连接用0.45μm聚醚砜过滤器至尖端。 <ol><li>添加在步骤2.6中收集的病毒介质插入注射器的筒，和沉浸介质为含有聚凝胺的新50mL锥形管中。通过真空抽吸从RD细胞除去培养基并过滤病毒介质添加到RD细胞。 </li></ol></li></ol> 第5天 <ol start="8"><li>重复第4天的HEK293T细胞可以在病毒媒体已经被收集之后被丢弃。 </li></ol> 7天 </STRONG&gt; <ol start="9"><li>洗涤RD细胞用1mL PBS中，并加入0.05％胰蛋白酶的200μL每孔。孵育在37℃下5分钟，添加补充培养基的2毫升，并移动细胞悬浮液到一个新的15毫升锥形。离心细胞，在250×g下在室温下并吸出介质5分钟。在1毫升DMEM中重悬（补充有5％FBS和1％青霉素 - 链霉素）。 注:使用FBS的比例较高可能导致流式细胞仪时堵塞<ol><li>过滤器的细胞用于流式细胞术。利用吸移管通过30微米的过滤器来应用1mL的RD细胞悬液到流式细胞术管和地方试管于冰上。 </li><li>排序EGFP +细胞26进入含有0.5毫升的DMEM的1.5mL微量离心管中补充有10％FBS和1％青霉素-链霉素和置于冰上。板EGFP +排序总共1mL的补充型DMEM中的细胞进入培养箱中培养12孔板和地点的单个孔中。 ;注意:EGFP +细胞从流这在转导后流式细胞仪的产量通常很低（50,000-100,000细胞）。细胞可能需要10培养 - 在进入步骤3之前14天。 </li></ol></li></ol> 3.的miR-200S的反向转染<ol><li>在-20℃下制备使用不含核酸酶的H 2只 O.分装股票和商店的miR-200模拟物的50μM股以避免反复冷冻/解冻循环。 </li><li>添加各为50μM的miR-200模拟物（的miR-200a中，的miR-200b中，和miR-200c中）至300μL无血清培养基或50μM阴性对照miRNA至300μL无血清培养基9μL的3μL。 </li><li>添加特异性siRNA转染试剂的6μL到无血清培养基的600μL和除以该混合物的300μL成两个管，一个用于每个来自步骤3.2所述两个的miR混合物。从步骤3.2的转染试剂的300μL的混合组合每个的miR混合物的300μL。孵育在室温TE 20分钟温度。 </li><li>而孵育，制备表达在第2节中2.4毫升创建EV或GRHL2 600000个EGFP + RD细胞的细胞悬浮液每治疗无血清培养基的（250个细胞/μL）。 </li><li>在24孔板中，每添加的miR-200的混合或来自步骤3.3阴性对照混合物的孔100μL成六个孔中，然后添加各细胞悬浮液的400μL到每个的miR混合物的三个孔。 </li><li>细胞孵育在37℃下在5％CO 2过夜和改变介质到完全补充的DMEM次日。收集单元2天后，使用合适的缓冲液（见下文）分析。经历MET RD肉瘤细胞的延时成像可作为补充材料。图像每个孔使用自动化的活细胞成像器27每2小时用10X物镜。 </li></ol> 4. RNA提取，逆转录，和qPCR <ol><li>根据使用标准RNA制造商的说明提取RNA提取试剂盒24。提取的RNA可被储存在-80℃。 </li><li>量化RNA浓度。解冻并与RNA冰工作。为了定量RNA浓度，根据制造商的方案在用酶标仪分光光度计260nm处测量UV吸光度。稀释所有样品与无核酸酶水的最低浓度。 </li><li>执行的总RNA逆转录成互补DNA（cDNA）。结合不低于100ng的总RNA用PCR缓冲液，dNTP混合物（100毫米），随机六聚体引，逆转录酶和无核酸酶的水在20μL反应体积24。根据制造商的协议运行RT周期。 cDNA可在-20℃保存长期。 </li><li>稀释20μL反应以100μL80μL无核酸酶的H 2 O的</li><li>混合5μL的基于荧光的嵌入染料2X的qPCR主混合物，各10μM的引物的0.06微升，一个ND 2μL每个样品稀释RT反应的。 </li><li>根据制造商的针对基于荧光的主混合物协议的qPCR运行。 </li><li>由增量CT方法量化mRNA的相对量和标准化为GAPDH。绘制生物学重复±每个治疗组的标准偏差的平均值mRNA的表达。 注:特殊的预防措施应与RNA时，以避免RNA酶，如使用无RNase的塑料和试剂接触服用。非一次性设备应使用去除RNA酶前处理。 </li></ol> 5.免疫荧光染色<ol><li>以下在24孔格式步骤3.6，吸媒体通过真空抽吸从RD细胞。 </li><li>固定细胞，加入每孔的4％多聚甲醛（PFA）的500μL，并孵育在室温下（RT）15分钟。固定后，位置细胞在磷酸盐在4℃下过夜如果需要缓冲盐水（PBS），并存储。 </li><li>通透细胞通过加入PBS + 0.2％的Triton-X100的500μL，并在室温下孵育30分钟。通过真空抽吸，除去透化缓冲液，并用PBS洗三次。 </li><li>在500微升的5％牛血清白蛋白的（BSA）/ PBS块孵育细胞在室温下30分钟。细胞可以在4℃保存过夜，如果必要的。 </li><li>准备一抗稀释。 /移液管将200μL的5％BSA的每孔PBS到15mL锥形（12个孔= 2.4毫升）和每毫升添加5％BSA的第一抗体1μL/ PBS需要。通过真空抽吸除去阻断缓冲液并分配稀释的一抗的200μL到每个孔中。 <ol><li>孵育细胞1:在5％BSA /在RT或过夜，在4℃1000稀释的初级抗体PBS 1个小时。温育后，洗涤细胞两次，用PBS。 </li></ol></li><li>在如上述5％BSA / PBS的相同体积的制备二级抗体稀释。添加使用1远红染料缀合的二级抗体:2000稀释。另外添加1微克/毫升Hoechst染料到混合的。取出PBS并添加混合200微升至每孔。 <ol><li>孵育在室温下在黑暗中1个小时。用PBS洗涤3次，在离开PBS的细胞，并使用铝箔保护细胞免受光。细胞如果需要，可以储存于4℃。 </li></ol></li><li>图像的细胞以400X总放大倍数与激发波长594倒置落射荧光显微镜 - 650纳米。 </li></ol> 6.免疫印迹<ol><li>用冰冷的PBS洗涤从3.6细胞两次。在冰上，用1x RIPA50μL的细胞裂解缓冲液补充有1X蛋白酶抑制剂混合物。 </li><li>在4℃下15分钟岩石裂解物，裂解物收集到1.5毫升管中，并离心以高速（20,000×g）离心5分钟以澄清的样品。细胞裂解物可在必要时被存储长期在-80℃下。 </li><li>量化使用Bradford测定总蛋白和蛋白质等分的等量到新的1.5毫升离心管中。布林克样本以等体积的使用RIPA缓冲液和3倍的Laemmli负载缓冲液补充有2-巯基乙醇。 </li><li>在95℃下孵育在1×Laemmli样品上样缓冲液的样品5分钟，并使用4-12％Tris-甘氨酸凝胶在200V下45分钟运行SDS-PAGE。取决于细胞系蛋白质装载范围的量。在一些情况下，需要的总蛋白的50-100微克检测间充质细胞系E-钙粘蛋白。 </li><li>转移蛋白到硝酸纤维素膜上2小时，在50 V在1×Tris-甘氨酸转移缓冲液。 </li><li>块膜在室温下或在4℃下在基于BSA阻断缓冲液中过夜1个小时。 </li><li>稀释的初级抗体在1:1000的浓​​度在5毫升封闭缓冲液中，加入稀释至膜，并在室温下或在4℃下过夜轻轻摇动1个小时孵育。在PBS中洗涤膜3次，用0.05％Tween-20的。 </li><li>在室温下孵育1个小时的膜与红外荧光偶联secondar在1 Y抗体:20,000稀释在如上述的封闭缓冲液。 </li><li>在PBS洗涤膜两次用0.05％吐温20，然后在一个时间PBS。 </li><li>使用标准的红外荧光检测图像的膜。 </li></ol> 7.锚地独立生长试验注:对于详细软琼脂测定方案，见28。 <ol><li>温暖的无菌2X DMEM培养基至42℃，在热水浴中。在此期间，热的预灭菌的1％琼脂糖在微波中3分钟。在根据需要或直至完全熔融的时间微波另外的30秒。琼脂糖转移到一个42℃的水浴中。 </li><li>放置一个50毫升锥形管中在无菌罩与42℃的水的烧杯中，并在1混合1％琼脂糖和2x DMEM培养基:1的比例占1.5毫升混合物，每孔在6孔板中。总是准备额外的DMEM /琼脂糖以弥补移液错误，避免气泡。 </li><li>加入混合物的的井侧，确保没有气泡形成，静置在室温下30分钟。 </li><li>而底层被固化，制备各组通过抽吸培养基并用1ml的PBS洗涤一次，在步骤3转染的RD细胞。吸PBS并加入0.2毫升0.05％胰蛋白酶并在37℃下孵育5分钟。 </li><li>在0.8毫升的培养基和磨碎的细胞中和胰蛋白酶，以形成单细胞悬浮液。转移细胞悬液至15mL锥形管中。 </li><li>计数细胞并计算每孔10,000个细胞所需的细胞悬浮液的体积。 </li><li>加热0.6％琼脂糖在微波中3分钟，接着30秒一次，直到完全熔化。琼脂糖转移到42℃水浴中。 </li><li>放置一个50毫升锥形管中在无菌罩与42℃的水的烧杯中。混合0.6％琼脂糖和稀释的细胞悬浮液以1:1的比例，以制备1.5毫升每孔的混合物在6孔板中。总是准备额外的细胞悬液/琼脂糖混合以弥补移液错误为了避免气泡。 注意:这是很重要的，在42℃这里工作。如果温度太低，则混合物将电镀之前固化，温度高于42℃会影响细胞生存力。 </li><li>通过研磨细胞几次拌匀细胞混合物添加到孔中，以确保无气泡的形式，并让固化在室温下20分钟。 </li><li> 加入2毫升补充培养基到每个孔中并移动到板孵化器。 <ol><li>更改媒体每周一次，3-4周或直至多细胞集落形成。要小心，以避免因负压吸引去除媒体接触时的琼脂。或者，使用P1000，以除去液体介质的顶层。 </li><li>加入200硝基μL蓝四氮唑氯化物溶液染色软琼脂平板上（1毫克/毫升在PBS中），并在37℃过夜孵育所述板。第二天，用PBS成像更换介质。 </li><li>图像在孔在倒置显微镜上40X总放大倍数。 </li><li>在ImageJ的执行分析菌落面积和数量。积平均生物学重复的集落数±标准偏差。 </li></ol></li></ol>

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作者什么都没有透露。

肉瘤是间充质细胞系的很少见，但高发性癌。尽管他们的间充质细胞系，肉瘤的一个子集似乎经历一个更上皮样状态的表型转化。这MET-像开关具有预后相关性，因为患者多上皮样的肿瘤较少侵略性24。尽管他们的临床相关性，很少有研究解决的分子机制肉瘤推动这些表型的转变。 为了检查肉瘤细胞MET样的转变，我们通过结合上皮因素GRHL2，且所述miR-200家族?...

An erratum was issued for: <a href="https://www.jove.com/video/55520/induction-of-mesenchymal-epithelial-transitions-in-sarcoma-cells">Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells</a>. An author name was updated.
One of the authors&#39; names was corrected from:
Shenghan Xu
to:
Shengnan Xu

An erratum was issued for: <a href="https://www.jove.com/video/55520/induction-of-mesenchymal-epithelial-transitions-in-sarcoma-cells">Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells</a>. An author name was updated.

Erratum: Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

可塑性是指细胞表型之间的可逆转变，并且通常分为两种类型，上皮 - 间质（EMT）的转变和间充质到上皮转变（MET）。此可塑性起着多细胞生物体，如发育和伤口愈合1的正常过程中起重要作用;然而，这些相同的途径和基因表达的方案也可导致疾病，如纤维化（评价的2，3，4）和癌转移（参考文献5，6，7审查，8）。期间转移，例如，EMT破坏细胞极性，细胞-细胞相互作用，并且促进侵袭9,10。总之，EMT贡献s至促进癌细胞的传播表型状态。此外，EMT也导致了驱动侵袭性表型的表型的其它改变，包括癌细胞代谢6的失调，药物抗性11,12的发展，增加的肿瘤引发能力13,14和宿主免疫逃避15的主机。 可塑性已经很好地研究了癌进展;然而，肉瘤还表现出表型可塑性。有趣的是，它看起来好像有些可塑性的癌相同的驱动程序也有助于肉瘤可塑性和侵略性。例如，从肉瘤患者的循环肿瘤细胞（CTC）已经显示出表达的EpCAM，是通常在上皮细胞上发现的16的细胞表面蛋白。阿迪倚重，250个软组织肉瘤样品被归类为上皮样或间充质样基于基因表达。在上皮样生物标记物标志患者比患者的间充质样生物标记物标志17预后较好。这与许多癌，在与患者多间充质样肿瘤18例相比，具有更多上皮样癌有更好的结果是一致的。 虽然一些肉瘤显示生物标记物和基因表达的途径与MET一致，此表型可塑性的分子基础仍然知之甚少。为了研究MET的机制和司机肉瘤我们开发使用两种特定的上皮细胞因子诱导MET的模型中，微小RNA（MIR）-200家庭和grainyhead样2（GRHL2）。与miR-200S是通过结合至MESSEN的3&#39; 非编码区调节基因表达的小的非编码RNA家族蒙古包RNA并防止翻译成蛋白质。与miR-200家族包括两个子组的 - 一种含的miR-141和miR-200a中，和另一种包括的miR-200b中，的miR-200c中，和miR-429。所述miR-200家族的成员富含上皮组织，和miR-200S的损失与转移癌19相关联。与miR-200家族也下调在软组织肉瘤与正常组织相比20。类似的miR-200S，GRHL2是上皮发展21重要的关键调节。所述GRHL2转录因子作用于两种方式上调上皮基因，如E-钙粘蛋白:1）在上皮细胞，GRHL2直接阻遏EMT主调节器，ZEB1 22;和2）GRHL2直接激活上皮基因23的转录。我们以前的研究已显示，在肉瘤细胞的miR-200和GRHL2的组合表达诱导MET样表型24。在这里，我们提出了一个详细的协议来创建肉瘤细胞使用的miR-200和GRHL2的异位表达MET诱导的体外模型。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Countess automated counter</td>
			<td>Life technologies</td>
			<td>AMQAX1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess cell counting chamber slides</td>
			<td>Invitrogen</td>
			<td>C10283</td>
			<td></td>
		</tr>
		<tr>
			<td>SimpliAmp Thermal Cycler</td>
			<td>Thermo Fisher</td>
			<td>A24811</td>
			<td></td>
		</tr>
		<tr>
			<td>Odyssey Fc</td>
			<td>LI-COR Inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ViiA7 Real Time PCR System</td>
			<td>Thermo Fisher</td>
			<td>4453536</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR microplate</td>
			<td>Corning</td>
			<td>321-29-051</td>
			<td></td>
		</tr>
		<tr>
			<td>KAPA SYBR Fast Universal qPCR Kit</td>
			<td>KAPA Biosystems</td>
			<td>KK4602</td>
			<td></td>
		</tr>
		<tr>
			<td>Starting Block (PBS) Blocking Buffer</td>
			<td>Thermo Fisher</td>
			<td>37538</td>
			<td>BSA-based blocking buffer</td>
		</tr>
		<tr>
			<td>Agarose General Purpose LE</td>
			<td>Genesee Scientific</td>
			<td>20-102</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris/Glycine/SDS Buffer</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>161-0732</td>
			<td>Running buffer</td>
		</tr>
		<tr>
			<td>10X Tris/Glycine Buffer</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>161-0734</td>
			<td>Transfer buffer</td>
		</tr>
		<tr>
			<td>RIPA Buffer</td>
			<td>Sigma Life Sciences</td>
			<td>SLBG8489</td>
			<td></td>
		</tr>
		<tr>
			<td>Amersham Protran 0.45 &#956;m nitrocellulose</td>
			<td>GE Healthcare Lifesciences</td>
			<td>10600012</td>
			<td></td>
		</tr>
		<tr>
			<td>Quick-RNA MiniPrep Kit</td>
			<td>Genesee Scientific</td>
			<td>11-358</td>
			<td></td>
		</tr>
		<tr>
			<td>Laemmli Sample Buffer (4X)</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>1610747</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Trans-Blot Cell</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>1703930</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-Protean Tetra Cell</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>1658005EDU</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Life technologies</td>
			<td>14190-144</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine RNAi Max</td>
			<td>Thermo Fisher</td>
			<td>13778150</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 Ragents</td>
			<td>Thermo Fisher</td>
			<td>11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Life technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>miRVana miRNA mimic negative control #1</td>
			<td>Thermo Fisher</td>
			<td>4464058</td>
			<td>neg miRNA</td>
		</tr>
		<tr>
			<td>hsa-miR-200 mirVana miRNA mimic</td>
			<td>Thermo Fisher</td>
			<td>4464066</td>
			<td>miR200A</td>
		</tr>
		<tr>
			<td>has-miR-200 mirVana miRNA mimic</td>
			<td>Thermo Fisher</td>
			<td>4404066</td>
			<td>miR200B</td>
		</tr>
		<tr>
			<td>has-miR-200 mirVana miRNA mimic</td>
			<td>Thermo Fisher</td>
			<td>4404066</td>
			<td>miR200C</td>
		</tr>
		<tr>
			<td>Opti-MEM</td>
			<td>Life technologies</td>
			<td>11088-021</td>
			<td>serum-free media</td>
		</tr>
		<tr>
			<td>anti-Ecadherin antibody</td>
			<td>BD Bioscience</td>
			<td>610182</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-beta actin</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-69879</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-EpCam</td>
			<td>Ab Serotec</td>
			<td>MCA18706</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-ZO1</td>
			<td>Invitrogen</td>
			<td>402200</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 800W</td>
			<td>LI-COR Inc</td>
			<td>925-32210</td>
			<td></td>
		</tr>
		<tr>
			<td>IRDye 680</td>
			<td>LI-COR Inc</td>
			<td>926-32223</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse AlexaFluor 647</td>
			<td>Thermo Fisher</td>
			<td>A211241</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit AlexaFluor 647</td>
			<td>Thermo Fisher</td>
			<td>ab150075</td>
			<td></td>
		</tr>
		<tr>
			<td>Halt Protease and Phosphatesse Inhibitor</td>
			<td>Thermo Fisher</td>
			<td>1861281</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color</td>
			<td>Bio-Rad Laboratories Inc</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>Partec CellTrics</td>
			<td>Sysmex</td>
			<td>04-004-2326</td>
			<td>30 &#956;m filter for flow</td>
		</tr>
		<tr>
			<td>GAPDH-F</td>
			<td>IDT</td>
			<td></td>
			<td>AGCCACATCGCTCAGACAC</td>
		</tr>
		<tr>
			<td>GAPDH-R</td>
			<td>IDT</td>
			<td></td>
			<td>GCCCAATACGACCAAATCC</td>
		</tr>
		<tr>
			<td>Ecadherin-F</td>
			<td>IDT</td>
			<td></td>
			<td>TGGAGGAATTCTTGCTTTGC</td>
		</tr>
		<tr>
			<td>Ecadherin-R</td>
			<td>IDT</td>
			<td></td>
			<td>CGCTCTCCTCCGAAGAAAC</td>
		</tr>
		<tr>
			<td>ZEB1-F</td>
			<td>IDT</td>
			<td></td>
			<td>GCATACAGAACCCAACTTGAACGTC</td>
		</tr>
		<tr>
			<td>ZEB1-R</td>
			<td>IDT</td>
			<td></td>
			<td>CGATTACACCCAGACTGC</td>
		</tr>
		<tr>
			<td>NOTCH-F</td>
			<td>IDT</td>
			<td></td>
			<td>GGCAATCCGAGGACTATGAG</td>
		</tr>
		<tr>
			<td>NOTCH-R</td>
			<td>IDT</td>
			<td></td>
			<td>CTCAGAACGCACTCGTTGAT</td>
		</tr>
		<tr>
			<td>nitro blue tetrazolium&#160;</td>
			<td>Sigma</td>
			<td>N5514</td>
			<td></td>
		</tr>
		<tr>
			<td>hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>polybrene</td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD Bioscience</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile syringe filter</td>
			<td>VWR</td>
			<td>28145-505</td>
			<td></td>
		</tr>
		<tr>
			<td>5mL polypropylene round-bottom tube</td>
			<td>352063</td>
			<td></td>
			<td>flow cytometry tubes</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Thermo Fisher</td>
			<td>4368814</td>
			<td>reverse transcription kit</td>
		</tr>
		<tr>
			<td>4% paraformaldyhyde</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281612</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X100</td>
			<td>Sigma</td>
			<td>93443</td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>A7906</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of mesenchymal-epithelial transitions in sarcoma cells

表型可塑性是指一种现象，其中细胞中瞬时获得另一谱系的性状。在癌的进展，表型可塑性驱动入侵，传播和转移。事实上，虽然大多数表型可塑性的研究一直在上皮来源的癌的情况下，原来的肉瘤，这是起源于间质，也表现出表型可塑性，有肉瘤的一个子集进行一个类似于间充质现象上皮转化（MET）。在这里，我们分别开发了一种方法，其包括与miR-200家族和grainyhead样2（GRHL2）以模仿肉瘤患者samples.We观察GRHL2和所述miR-200家族使用细胞转导和转染顺序表达这种MET-状现象， ，以更好地理解肉瘤细胞表型，这些转变的分子基础。表达的miR-200和GRHL2肉瘤细胞显示增强的上皮characterist集成电路细胞形态和上皮和间充质标志物的改变。使用这些方法今后的研究可以被用来更好地了解肉瘤细胞MET状突起，如迁移，侵入，转移倾向，和治疗的抗性的表型的影响。

架构MET诱导细胞肉瘤对于在肉瘤细胞MET样改变感应的一般时间线示于图1。该协议开始于转导GRHL2（ 图1A），随后所述miR-200家族（ 图1B）的转染。 GRHL2或miR-200家族成员未能单独表达时冲击RD细胞的出现，但GRHL2和miR-200S的异位表达一起导致在RD细胞上皮样形态学变化。从一个纺锤状的形式，以?...

Watch this Scientific Journal Video about Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells at JoVE.com

Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

我们在这里提出用于诱导基于微小RNA-200家族成员及grainyhead样2（GRHL2）的合并的异位表达肉瘤细胞间质上皮转换（MET）的细胞培养方法。这种方法适用于更好地理解可塑性的癌症侵袭性和治疗方法的生物学影响。

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 ...

induction-of-mesenchymal-epithelial-transitions-in-sarcoma-cells

Research

JoVE Journal

Developmental Biology

9.3K 次观看.  Duke University. 我们在这里提出用于诱导基于微小RNA-200家族成员及grainyhead样2（GRHL2）的合并的异位表达肉瘤细胞间质上皮转换（MET）的细胞培养方法。这种方法适用于更好地理解可塑性的癌症侵袭性和治疗方法的生物学影响。 

视频: Induction of Mesenchymal-Epithelial Transitions in Sarcoma Cells

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

脂肪干细胞的软骨细胞分化诱导离心重力

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

人多能干细胞对原始和最终造血祖细胞定向分化的研究

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

诱导生活青蛙和斑马鱼胚胎缺氧

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Horizontal Whole Mount: A Novel Processing and Imaging Protocol for Thick, Three-dimensional Tissue Cross-sections of Skin

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality microscopic images is not entirely dependent upon the quality of the microscope, but also upon the methods of tissue processing, which often involve multiple critical actions or steps. Furthermore, mesenchymal cell types in the skin and other tissues represent a new challenge for tissue preparation and imaging. Here, we present a complete process, from tissue harvest to microscopy. Our technique, called &#34;horizontal whole mount,&#34; is one that novices can quickly become proficient in and that allows for antigen preservation and detection in 60-300 &#181;m-thick sections cut with a cryostat. Sections of this thickness provide enhanced visualization of tissue microarchitecture in a three-dimensional environment. In addition, the protocol preserves mesenchymal cells in a manner that enhances image quality when compared to standard cryostat or paraffin sections, thereby increasing the efficacy and reliability of immunostaining. We believe that this protocol will benefit all laboratories that visualize skin, and possibly other tissues and organs.

This work presents a novel processing and imaging protocol for thick, three-dimensional tissue cross-section analysis that enables the full exploitation of confocal imaging modalities. This protocol preserves antigenicity and represents a robust system to analyze skin histology and potentially other tissue types.

水平整体安装:厚三维组织横截面皮肤的新型加工和成像方案

Processing a tissue of interest to generate a microscopic image that supports a scientific argument can be challenging. The acquisition of high-quality ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

活完好斑马鱼的单细胞 Photoconversion

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

低血清条件下心脏祖细胞内皮分化的诱导作用

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...