这项工作得到了瑞士国家科学基金会 Sinergia 赠款 CRSII3 147697, 基金会《应用 Krebsforschung 和苏黎世 Krebsliga 的支持。

1. 胞浆和核蛋白分数的制备
<ol><li>板 4 x 106 ACC-MESO-4 细胞在 T150 细胞培养瓶中生长 RPMI 1640 培养基补充10% 血清, 1x 青霉素/链霉素 (100x), 2 毫米 l-谷氨酰胺 (200 毫米)。当细胞达到 80-90% (~ 5-5. 5 x 106细胞) 的汇合处时, 继续进行蛋白质提取核和胞浆分数。 注: ACC-MESO-4 细胞线是从山本理 BioResource 中心11获得的。</li>
	<li>吸入培养基, 通过加入15毫升的 1x PBS 来洗涤细胞, 轻轻地将盘子倾斜几次, 吸入 1x PBS。添加3毫升0.25% 胰蛋白酶-EDTA 和孵化培养瓶在37°c 3 分钟。 注: 观察显微镜下的细胞脱离;如果仍然附有水龙头对手手掌3次。</li>
	<li>添加7毫升的 RPMI-1640 培养基, 辅以10% 的血清, 1x 青霉素/链霉素 (100x), 2 毫米 l-谷氨酰胺 (200 毫米), 收集的细胞在15毫升管, 并在 200 x g 向下旋转5分钟。</li>
	<li>吸入培养基和并用重悬细胞颗粒, 1.5 毫升 1x PBS, 然后将细胞转化为1.5 毫升离心管。向下旋转 500 x g 和4°c 为5分钟, 丢弃上清。 注意: 从这个步骤开始, 执行所有离心步骤在4摄氏度, 并保持所有试剂在冰上。</li>
	<li>并用重悬的细胞颗粒与冰冷1.5 毫升 1x PBS 和旋转向下的细胞在 500 x g 和4°c 3 分钟. 丢弃上清。 注: 所需量的 cer I、cer II 和净净试剂分别为10、0.55 和5卷的估计细胞颗粒包装量;下面的协议假定容量为20µL。我们建议只准备所需量的试剂 CER i。</li>
	<li>添加200µL 的冰冷细胞质萃取试剂 (CER I) 补充2µL 的1x 蛋白酶抑制剂,例如, 稀释1片蛋白酶抑制剂 (无 EDTA) 在500µL 的水获得100x 的库存。这一量的试剂 CER 我足够溶解20µL 细胞颗粒包装体积。<ol>
<li>要估计细胞颗粒的包装体积, 比较的管含有细胞颗粒与1.5 毫升管填充 10, 20, 50 或100µL 1x PBS。</li>
	</ol>
</li>
	<li>并用重悬细胞颗粒通过大力涡流管十五年代和孵化的管在冰上10分钟. 加入11µL 的冰冷细胞质萃取试剂 ii (CER ii) 到管, 大力漩涡 5 s, 并孵化1分钟冰。</li>
	<li>漩涡大力为 5 s 和离心机管在 1.6万 x g 和4°c 5 分钟. 将上清液转移到冰上干净的预冷管上。上清是在 RNA 下拉实验中进一步使用的细胞质分数。</li>
	<li>并用重悬100µL 冷核萃取试剂中剩余的颗粒, 补充1µL 蛋白酶抑制剂 (100x) 和大力涡十五年代。我们建议只准备所需量的净净试剂补充蛋白酶抑制剂。</li>
	<li>在冰上孵化样品40分钟, 偶尔涡流十五年代 (每10分钟)。</li>
	<li>离心管在 1.6万 x g 10 分钟转移上清 (这是核蛋白分数) 到一个干净的预冷管。</li>
	<li>立即通过使用二辛可宁 (评估) 方法测量蛋白质浓度 (参见材料表) 来进行。为了控制每个分数的蛋白质纯度, 执行免疫印迹法(图 1) 对α蛋白 (仅在胞浆分数中的阳性检测) 和聚 ADP-核糖聚合酶-PARP (仅在核分数中进行阳性检测)。对于蛋白质可视化, 请参阅第4节。</li>
	<li>为了保持蛋白质的活性和其自身的状态, 准备25µL 的整除数胞浆和核萃取物。把这些整除数放在液氮中5秒, 然后直接储存在-80 摄氏度。</li>
</ol>2. 用 Desthiobiotin 标记 RNA
<ol><li>有商业合成的 RNA 探针和高效液相色谱纯化。并用重悬与核酸酶水在10µM。 注意: 探测序列列在表 1中。</li>
	<li>每个 rna 下拉反应使用 50 pmol rna。解冻并保持所有的试剂在冰上, 除了 PEG 30%, 用来标记 3 ' 的 RNA 寡核苷酸的终点。</li>
	<li>转移5µL 10 µM RNA 探针, 标记为 CALB2 3 ' UTR (是), CALB2 3 ' UTR (mtARE) 和不相关的 RNA (愤怒), 入0.5 毫升薄壁离心管并且孵化在 PCR 机在85°c 为5分钟. 立即将管子放在冰上。 注意: 这一步很重要, 因为它促进了 RNA 探针的松弛和可访问性以进行标记。</li>
	<li>对于单一的30µL 反应, 添加到含 RNA 管10µL 混合: 3 µL 核酸酶游离水, 3 µL 10x RNA 连接酶反应缓冲器, 1 µL RNase 抑制剂 (40 U/µL), 1 µL Desthiobiotinylated 胞苷 Bisphosphate (1 毫米)和2µL T4 RNA 连接酶 (20 U/µL)。 注: 要准备3个样本超过 (3.2) 的主混合 A, 混合9.6 µL 的核酸酶游离水, 9.6 µL 的 10x RNA 连接酶反应缓冲器, 3.2 µL RNase 抑制剂 (40 U/µL), 3.2 µL 的 Desthiobiotinylated 胞苷 Bisphosphate (1 毫米), 6.4 µL T4 RNA 连接酶 (20 U/µL)。</li>
	<li>仔细添加15µL 的 PEG 30% 的反应。使用另一个提示混合反应。在16摄氏度一夜之间孵化反应。</li>
	<li>第二天, 准备以下内容: 5 米氯化钠 (新鲜/核酸酶), 氯仿: 异戊醇在24:1 比率 (例如, 每反应-96 µL 氯仿和4µL 异戊醇), 冰冷100% 乙醇和冰冷70% 乙醇.</li>
	<li>添加70µL 的核酸酶水到 RNA 标记反应管。添加100µL 氯仿: 异戊醇和涡旋简要。在 1.3万 x g 的旋转向下3分钟小心地移除上部阶段和转移到一个新的无核酸酶 1.5 mL 管;避免接触较低的相位。</li>
	<li>添加10µL 5 米氯化钠, 1 µL 糖原和300µL 冰冷100% 乙醇。把管子放在-20 摄氏度, 2 小时。 注: 在这一步, 实验可以继续在第二天。</li>
	<li>离心机在 1.3万 x g 和4°c 为15分钟, 小心地丢弃上清, 不干扰颗粒。300µL 70% 乙醇和离心机再次在 1.3万 x g 和4°c 为5分钟。</li>
	<li>完全丢弃上清液, 并风干颗粒 (15 分钟)。并用重悬20µL 的核酸酶水中的颗粒。 注意: 在同一天进行 RNA 下拉。</li>
	<li>孵育 RNA 在90°c 2 分钟和地方在冰。在链亲和素磁性微珠预洗涤的以下步骤中, 将标记 RNA 放在冰上。 注意: 长时间潜伏期可能会损害 RNA 探针。</li>
</ol>3. RNA 蛋白质下拉
<ol><li>
预洗链亲和素磁性微珠与 desthiobiotin RNA 孵化
	<ol>
<li>每 50 pmol RNA 使用50µL 链亲和素磁性珠子。但是, 这应该根据实验进行优化。</li>
		<li>涡流管与链亲和素磁珠十五年代, 并迅速删除200µL (主混合; 足够的 3 + 1 反应) 成一个干净的1.5 毫升安全锁管使用切割吸管提示。把管子放在磁性支架上, 这样珠子就会在管子的侧面收集, 等待1分钟. 取出泥沙液体。</li>
		<li>要清洗珠子, 从磁支架上取下管子, 加入400µL 0.1 米氢氧化钠, 0.05 米氯化钠溶液, 轻轻吸管上下几次。把管子放回磁性支架上。等1分钟, 收集上清。再次重复此步骤。</li>
		<li>用200µL 100 毫米氯化钠冲洗珠子。移除上清。</li>
		<li>添加200µL 20 毫米三 (pH 7.5), 并用重悬珠吹打, 放置在磁性立场的管, 等待1分钟, 并删除上清。重复步骤。</li>
		<li>将管子从磁支架上取下, 加入200µL 1x RNA 捕获缓冲器, 并用重悬链亲和素磁性微珠涡流。去除50µL 的链亲和素磁性珠子, 并添加到每个标记 RNA 管使用切割吸管尖端。在滚筒上室温下孵化管30分钟。</li>
	</ol>
</li>
	<li>
蛋白质与 RNA 的结合
	<ol>
<li>将管子放进磁性支架, 等待1分钟, 取出上清。</li>
		<li>添加50µL 20 毫米三 (pH 7.5) 的珠子和并用重悬由吹打。把管子放到磁性支架上, 等待1分钟, 取出上清。重复此步骤。</li>
		<li>添加100µL 1x 蛋白-RNA 结合缓冲剂的珠子和并用重悬由吹打。</li>
		<li>同时, 准备以下组合:10 µL 10x 蛋白-RNA 结合缓冲液, 30 µL 50% 甘油, 胞浆蛋白50µg, 无核酸酶水可达100µL。 注: 准备主混合 B 为3个样品过剩 (3.3) 如下:33 µL 10x 蛋白-RNA 结合缓冲, 99 µL 50% 甘油, 165 µg 细胞质蛋白和核酸酶水多达330µL. 把管子的主混合 B 放在冰上。</li>
		<li>把管子放到磁性支架上, 等待1分钟, 然后收集上清。添加100µL 的主混合 B, 并用重悬轻轻吹打上下, 并避免产生气泡。孵化反应管为60分钟在4°c 在滚筒上。</li>
	</ol>
</li>
	<li>
洗涤和洗脱
	<ol>
<li>把管子放到一个磁性的支架上, 收集上清液 (流经 FT), 然后转移到新的管子里。 注意: 将此 FT 放在冰上以供以后分析。</li>
		<li>吸管100µL 的1x 洗涤缓冲器上的珠子, 轻轻并用重悬, 放回磁性支架, 等待1分钟, 并丢弃上清。重复此步骤2次。</li>
		<li>在磁珠上添加40µL 洗脱缓冲器, 混合吹打上下, 并在37摄氏度上孵化在管振动筛 (950 rpm) 30 分钟。</li>
		<li>将管子放入磁性支架, 等待1分钟, 收集洗脱样品 (洗脱液)。放置在冰上, 用于下游分析。 注意: 在这个步骤中, 每个经过测试的 RNA 探针都由一个流经 (FT) 和洗脱液 (E) 样本组成。</li>
	</ol>
</li>
</ol>4. 聚丙烯酰胺电泳与印迹分析
注: 有关本节中使用的缓冲区的食谱, 请参见表2。根据 Laemmli 方法12执行西方污点。
<ol><li>Handcast 12% SDS 页凝胶 (10 井, 1.5 毫米间隔, 66 µL)。准备一个运行的凝胶混合: 4 毫升30% 丙烯酰胺-bisacrylamide 库存溶液, 2.4 毫升下三缓冲器, 3.2 毫升的 dH2O, 9.6 µL TEMED, 96 µL 10% 硫酸铵溶液 (APS)。准备堆叠凝胶混合: 3.25 毫升 dH2O, 0.5 毫升30% 丙烯酰胺-bisacrylamide 库存溶液, 上三个缓冲器1.3 毫升, 10 µL TEMED, 20 µL 10% APS。</li>
	<li>添加16.6 µL 6x Laemmli 缓冲区 (例如, 14 毫升的三/HCl/sds pH 值 6.8 (上部三缓冲器), 2 克的 sds, 1.86 克的德勤, 6 毫升甘油和0.012% 溴酚蓝色; 存储在-20 °c) 到 FT 样本, 6.6 µL 6x Laemmli 缓冲区进入 e 样品。孵育样品5分钟, 在95摄氏度。</li>
	<li>装载样品 (20 µL 的 FT 和整个洗脱 ~ 40 µL) 和运行他们30分钟在 60 v, 继续与 20 V 隔夜。</li>
	<li>使用半干 electroblotting 系统, 将蛋白质转移到 PVDF 膜上80分钟, 在 15 v。 注: 在执行蛋白质转移之前, 必须激活 PVDF 膜。将膜在100% 甲醇中孵化1分钟, 然后在超纯水中孵化2分钟。</li>
	<li>蛋白质转移后, 用考马斯溶液孵化 SDS 页凝胶3小时, 然后用超纯水洗涤三步, 每30分钟。</li>
	<li>在蛋白质转移结束时, 让 PVDF 膜干燥1小时, 如步骤4.5 所示, 重新激活膜。在 1x TTBS 中用 5% BSA 阻挡膜1小时。</li>
	<li>用第一个抗体孵化膜: 虚宾或间皮素, 在室温下或一夜4摄氏度稀释1:1000 在 5% BSA 在 1x TTBS 4h。</li>
	<li>在 1x TTBS 中冲洗三次, 用兔抗小鼠辣根过氧化物酶 (HRP)-共轭次生抗体稀释 1:10,000 5% BSA, 1x TTBS。</li>
	<li>使用增强化学发光和数字成像仪可视化蛋白质。</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+of+regulatory+RNA.">The rise of regulatory RNA.</a> Nature Reviews: Genetics. 15 (6), 423-437 (2014).</li><li>Rinn, J. 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=Functional+demarcation+of+active+and+silent+chromatin+domains+in+human+HOX+loci+by+noncoding+RNAs.">Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.</a> Cell. 129 (7), 1311-1323 (2007).</li><li>Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+proteins+targeted+by+a+short+repeat+RNA+to+the+mouse+X+chromosome.">Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome.</a> Science. 322 (5902), 750-756 (2008).</li><li>McHugh, C. 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=The+Xist+lncRNA+interacts+directly+with+SHARP+to+silence+transcription+through+HDAC3.">The Xist lncRNA interacts directly with SHARP to silence transcription through HDAC3.</a> Nature. 521 (7551), 232-236 (2015).</li></ol>

作者声明他们没有竞争的财政利益。

3 ' UTRs 属于非编码基因组3, 所有非编码 rna 可以与蛋白质相互作用, 以发挥其功能7。当哺乳动物基因组被发现被普遍转录并产生长非编码 rna 16 的很大一部分时, 新的证据表明这些长非编码 rna 在调节基因表达时的作用与染色质重塑复合体17。这种知识是利用 RNA 下拉法得到的。因此, 要开始破译特定 RNA 的 interactors, 第一步可能?...

基因表达和基因产物的最终水平可以通过影响 mrna 稳定性和 mrna 翻译率1来严格调节。这些转录后调控机制是通过非编码 rna 和/或 rna 结合蛋白 (限制性商业惯例) 与靶向 mRNA 的相互作用而施加的。它通常是 3 ' 未翻译的 mRNA 区域 (3 ' UTR-属于基因组2) 的非编码部分, 其中包含特定的cis-管理元素 (), 由反演作用因素 (如 miRNA 或限制性商业惯例) 识别。3. 最好研究的cis元素在 3 ' UTR, 是腺嘌呤丰富的元素 (是) 的主题, 这是公认的特定的 AU 结合蛋白 (AUBP), 反过来, 诱导 mRNA 退化/deadenylation (是介导的衰变) 或mRNA 稳定化4。
3 ' UTR 的 calretinin mRNA (CALB2) 的大小是 573 bp 长, 包含一个假定的 AUUUA pentamer, 如 AREsite2 的预测, 生物信息学工具 5.与假定的存在相一致的是主题, pmirGLO 向量-报告分析显示了这个元素在 CALB2 mRNA6中的稳定作用。最后, 用体外RNA 下拉法确定了通过 AUBP 稳定 calretinin mRNA 的方法。
由于所有非编码 rna 与蛋白质7相互作用,体外rna 下拉是确定 rna interactors 以帮助破译分子机制的一种很好的方法和首选的检测手段。在这个 rna 下拉法, 商业上被合成的 rna 探针, 被标记以被改进的生物素 (desthiobiotin) 的形式在 RNA 链的 3 ' 末端被使用了。rna desthiobiotin 是固定的通过与链亲和素的相互作用的磁性珠子, 这是用来拉下来的蛋白质, 特别是与绑定 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。这种 RNA 束缚的蛋白质是洗脱从磁性珠子, 运行通过 12% SDS 页凝胶, 转移到膜, 并探讨了不同的抗体。
在标准的链亲和素-生物素亲和纯化过程中, 需要苛刻的变性条件来破坏强不可逆转的生物素-链亲和素键洗脱绑定蛋白 8, 这可能导致离解蛋白质复合物。与生物素不同的是, desthiobiotin 可逆地结合到链亲和素, 并以缓冲的生物素溶液进行竞争性置换, 允许蛋白质的温和洗脱, 避免自然生物素化分子的分离 9,表明该技术是理想的分离本地蛋白质复合体在当地条件下。
体外约束条件和严格性是由盐浓度、还原剂和洗涤剂百分比决定的, 应与细胞上下文中存在的情况相近, 以便识别真正的体内交互作用。在此之前所实现的绑定条件已被证明是适当的, 以确定虚宾作为一种金结合蛋白10。这种方法可以节省时间, 因为优化适当的绑定条件可能会耗费时间和挑战性。此外, 该方法可作为任何 RNA 下拉实验的启动协议, 通过改变盐和洗涤剂的浓度, 改变甘油的百分比, 添加其他盐, 可以逐步优化。此外, 我们证明, 即使是一个短的25核苷酸 RNA 探针窝藏 pentamer 是-主题可以用来证明与特定 AUBP 的互动。

<table border="0" cellpadding="0" cellspacing="0" width="1340">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">NE-PER Nuclear and cytoplasmic extraction kit</td>
			<td style="width:295px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:124px;">78833</td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Protease Inhibitor Tablets, EDTA-free</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:124px;">A32965</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce Magnetic RNA-protein Pull-Down Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20164</td>
			<td>Includes magnetic beads and therefore the kit need to be stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pierce RNA 3&#8217; End Desthiobiotinylation Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20163</td>
			<td style="width:468px;">The kit is a part of the &#34;Pierce Magnetic RNA-protein Pull-Down Kit&#34;, and needs to be stored at -20 &#176;C unlike the pull-down kit (4 &#176;C).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2,&#160; magnetic stand</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">12321D</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:453px;">Anti - HuR (MOUSE) monoclonal antibody</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">-</td>
			<td style="width:468px;">The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&#160; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - &#945; - tubulin (MOUSE) monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td style="width:124px;">8035</td>
			<td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - PARP (RABBIT) Polyclonal antibody</td>
			<td>Cell Signaling</td>
			<td>9542</td>
			<td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - Mesothelin (MOUSE) Monoclonal Antibody&#160;</td>
			<td>Rockland</td>
			<td>200-301-A87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary goat anti-rabbit antibody</td>
			<td style="width:295px;">Cell Signaling</td>
			<td style="width:124px;">7074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary rabbit anti-mouse antibody</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A-9044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RPMI - 1640 medium</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FBS - Filtrated Bovine Serum</td>
			<td style="width:295px;">Pan Biotech&#160;</td>
			<td style="width:124px;">P40-37500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin - streptomycin (100x)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">L-Glutamine solution (200 mM)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">0.25% Trypsin-EDTA (1x)</td>
			<td style="width:295px;">Gibco by Life technologies&#160;</td>
			<td style="width:124px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN 3 Cell</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini Protean System Glass Plates&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &#181;L</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra Cell Casting Modules</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1658050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNaseZap RNase Decontamination Solution</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:453px;">Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td>
			<td style="width:295px;">Roth</td>
			<td style="width:124px;">3029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">20% SDS&#160;</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td>A3942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">PVDF transfer membrane&#160;</td>
			<td style="width:295px;">Perkin Elmer</td>
			<td style="width:124px;">NEF1002</td>
			<td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Trans-Blot SD semi-dry transfer cell&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Clarity Western ECL Substrate</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1705061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FusionFX&#160; Digital Imager</td>
			<td style="width:295px;">Vilber</td>
			<td style="width:124px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNA oligo synthesis</td>
			<td style="width:295px;">Mycrosynth AG, Switzerland</td>
			<td style="width:124px;">-</td>
			<td>the synthesis scale - 0.04 &#181;mol - HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Bovine serum albumin</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Hydrochloric Acid (HCl) 6 M</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td style="width:124px;">182883.1211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ultra Pure 1 M Tris, pH 7.5&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Glycerol&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td>17904</td>
			<td>50% sterile aliquots to be stored at -20&#176;C</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:453px;">Pierce Streptavidin magnetic beads&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">88817</td>
			<td style="width:468px;">Please note that these magnetic beads have an average diameter of 1 &#181;m (range from 0.5 - 1.5 &#181;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &#181;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">TEMED</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ammonium persulfate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coomassie G-250</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A3480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminiumsulfate-(14-18)-hydrate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">368458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ortho-phosphoric acid&#160;</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A0637</td>
			<td></td>
		</tr>
	</tbody>
</table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

体外RNA 下拉仍然主要用于确定 rna 结合蛋白识别特定 rna 结构和图案的协议的第一步。在这个 rna 下拉协议, 商业上合成的 rna 探针被标记以修改的形式生物素, desthiobiotin 在 RNA 链的 3 ' 末端, 可逆地束缚到链亲和素并且因而允许蛋白质洗脱在更加生理上条件。rna desthiobiotin 是固定的, 通过与链亲和素的相互作用, 磁性珠子, 这是用来拉下来的蛋白质, 特别是与 RNA 的利益。非变性和活性蛋白从间皮瘤细胞胞浆的比例被用作蛋白质的来源。此处描述的方法可用于检测已知的 rna 结合蛋白与含有一系列兴趣序列的25核苷酸 (nt) 长 RNA 探针之间的相互作用。这对于完成使用报告向量法实现的 RNA 分子中稳定或不稳定元素的功能表征是有用的。

在这个实验中, 一个 25 nt 长片段的 calretinin 3 ' UTR 窝藏是主题 (CALB2 3 ' UTR (是) 25 nt) 被用来测试它是否结合了人类抗原 R (虚) 蛋白, 一个已知的 mRNA 稳定剂。为了测试这些元素的特异性, 25-nt RNA 探针 CALB2 3 ' UTR (mtARE) 包含了一个有主题的突变, 以前被证明是为了取消 "是" 的主题的稳定效果, 使用了6。第三个 RNA 探针代表负控制, 这是一个 28 nt 无关的 rna, 它包...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-链亲和素亲和介导纯化间皮瘤细胞 RNA 相互作用蛋白的研究

Desthiobiotin 标记的合成 25-核苷酸 RNA 寡核苷酸, 其中含有腺嘌呤丰富的元素 (是) 的主题, 允许特定的结合胞浆是结合蛋白。

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

desthiobiotin-streptavidin-affinity-mediated-purification-rna

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

7.9K 次观看.  University Hospital Zurich. Desthiobiotin 标记的合成 25-核苷酸 RNA 寡核苷酸, 其中含有腺嘌呤丰富的元素 (是) 的主题, 允许特定的结合胞浆是结合蛋白。

视频: Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.

The uneven loss of medium from microtiter plates affects the reproducibility of uniform multicellular tumor spheroid formation. Improving culture conditions to reduce significant medium loss will improve the reproducibility of spheroid formation and the results of spheroid-based assays using the liquid-overlay technique.

蒸发减少培养条件的提高多细胞球体形成的微孔板重现

Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential ...

科研

癌症研究

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying cancer metastasis in vivo, other efficient, reliable, low-cost models are needed to quickly access the potential effects of (epi)genetic changes or pharmacological compounds. As such, we illustrate and explain the feasibility of xenograft models using human breast cancer cells injected into zebrafish embryos to support this goal. Under the microscope, fluorescent proteins or chemically labeled human breast cancer cells are transplanted into transgenic zebrafish embryos, Tg (fli:EGFP), at the perivitelline space or duct of Cuvier (Doc) 48 h after fertilization. Shortly afterwards, the temporal-spatial process of cancer cell invasion, dissemination, and metastasis in the living fish body is visualized under a fluorescent microscope. The models using different injection sites, i.e., perivitelline space or Doc are complementary to one another, reflecting the early stage (intravasation step) and late stage (extravasation step) of the multistep metastatic cascade of events. Moreover, peritumoral and intratumoral angiogenesis can be observed with the injection into the perivitelline space. The entire experimental period is no more than 8 days. These two models combine cell labeling, micro-transplantation, and fluorescence imaging techniques, enabling the rapid evaluation of cancer metastasis in response to genetic and pharmacological manipulations.

Here, we describe xenograft zebrafish models using two different injection sites, i.e., perivitelline space and duct of Cuvier, to investigate the invasive behavior and to assess the intravasation and extravasation potential of human breast cancer cells, respectively.

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average result masking the rich single cell behavior within a complex population. Single cell protein detection and quantification technologies have made a remarkable impact in recent years. Here we describe a practical and flexible single cell analysis platform based on addressable droplet microarrays. This study describes how the absolute copy numbers of target proteins may be measured with single cell resolution. The tumor suppressor p53 is the most commonly mutated gene in human cancer, with more than 50% of total cancer cases exhibiting a non-healthy p53 expression pattern. The protocol describes steps to create 10 nL droplets within which single human cancer cells are isolated and the copy number of p53 protein is measured with single molecule resolution to precisely determine the variability in expression. The method may be applied to any cell type including primary material to determine the absolute copy number of any target proteins of interest.

Here we present addressable droplet microarrays (ADMs), a droplet array based method able to determine absolute protein abundance in single cells. We demonstrate the capability of ADMs to characterize the heterogeneity in expression of the tumor suppressor protein p53 in a human cancer cell line.

用可寻址滴微阵列计算单细胞中的蛋白质

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

骨肉瘤前小鼠模型对肿瘤与间充质干细胞细胞外囊泡介导通信的定义

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter assays, embryonic stem cell viability assays, and therapeutic drug-based sensitivity assays. Although they have clarified a lot of BRCA1 variants, these assays involving the use of exogenously expressed BRCA1 variants are associated with overexpression issues and cannot be applied to post-transcriptional regulation. To resolve these limitations, we previously reported a method for functional analysis of BRCA1 variants via CRISPR-mediated cytosine base editor that induce targeted nucleotide substitution in living cells. Using this method, we identified variants whose functions remain ambiguous, including c.-97C&#62;T, c.154C&#62;T, c.3847C&#62;T, c.5056C&#62;T, and c.4986+5G&#62;A, and confirmed that CRISPR-mediated base editors are useful tools for reclassifying the variants of uncertain significance in BRCA1. Here, we describe a protocol for functional analysis of BRCA1 variants using CRISPR-based cytosine base editor. This protocol provides guidelines for the selection of target sites, functional analysis and evaluation of BRCA1 variants.

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

People with BRCA1 mutations have a higher risk of developing cancer, which warrants accurate evaluation of the function of BRCA1 variants. Herein, we described a protocol for functional assessment of BRCA1 variants using CRISPR-mediated cytosine base editors that enable targeted C:G to T:A conversion in living cells.

使用 CRISPR 介质基础编辑器对 BRCA1 变体进行功能评估

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as drug resistance, epithelial-mesenchymal transition (EMT), and metastasis. Intratumoral eATP is 103 to 104 times higher in concentration than in normal tissues. While eATP functions as a messenger to activate purinergic signaling for EMT induction, it is also internalized by cancer cells through upregulated macropinocytosis, a specific type of endocytosis, to perform a wide variety of biological functions. These functions include providing energy to ATP-requiring biochemical reactions, donating phosphate groups during signal transduction, and facilitating or accelerating gene expression as a transcriptional cofactor. ATP is readily available, and its study in cancer and other fields will undoubtedly increase. However, eATP study remains at an early stage, and unresolved questions remain unanswered before the important and versatile activities played by eATP and internalized intracellular ATP can be fully unraveled.
These authors&#39; laboratories&#39; contributions to these early eATP studies include microscopic imaging of non-hydrolysable fluorescent ATP, coupled with high- and low-molecular weight fluorescent dextrans, which serve as macropinocytosis and endocytosis tracers, as well as various endocytosis inhibitors, to monitor and characterize the eATP internalization process. This imaging modality was applied to tumor cell lines and to immunodeficient mice, xenografted with human cancer tumors, to study eATP internalization in vitro and in vivo. This paper describes these in vitro and in vivo protocols, with an emphasis on modifying and finetuning assay conditions so that the macropinocytosis-/endocytosis-mediated eATP internalization assays can be successfully performed in different systems.

We developed a reproducible method to visualize the internalization of nonhydrolyzable fluorescent adenosine triphosphate (ATP), an ATP surrogate, with high cellular resolution. We validated our method using independent in vitro and in vivo assays-human tumor cell lines and immunodeficient mice xenografted with human tumor tissue.

荧光显微镜用于人肿瘤细胞和肿瘤异种移植小鼠中巨细胞增多介导的ATP内化

Adenosine triphosphate (ATP), including extracellular ATP (eATP), has been shown to play significant roles in various aspects of tumorigenesis, such as ...

Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary tumor cells from surgically resected, malignant pleural mesothelioma (MPM) by providing a complete overview of the process from digestion to enrichment, expansion, cryopreservation, and phenotypic characterization. In addition, this protocol introduces concepts for tumor generation that may be useful for multiple tumor types such as differential trypsinization and the impact of dissociation methods on the detection of cell surface markers for phenotypic characterization. The major limitation of this study is the selection of tumor cells that will expand in a two-dimensional (2D) culture system. Variations to this protocol, including three-dimensional (3D) culture systems, media supplements, plate coating to improve adhesion, and alternate disaggregation methods, could improve this technique and the overall success rate of establishing a tumor line. Overall, this protocol provides a base method for establishing and characterizing tumor cells from this rare tumor.

The goal of this method paper is to demonstrate a robust and reproducible methodology for the enrichment, generation, and expansion of primary tumor cell lines from surgically resected pleural mesothelioma.

原发性恶性胸膜间皮瘤肿瘤系的生成和扩展

Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites have been the gold standards for conditional or inducible gene targeting. To provide faster and more efficient experimental models, an ex vivo organoid culture system is developed using adenovirus Cre and normal urothelial cells carrying multiple loxP alleles of the tumor suppressors Trp53, Pten, and Rb1. Normal urothelial cells are enzymatically disassociated from four bladders of triple floxed mice (Trp53f/f: Ptenf/f: Rb1f/f). The urothelial cells are transduced ex vivo with adenovirus-Cre driven by a CMV promoter (Ad5CMVCre). The transduced bladder organoids are cultured, propagated, and characterized in vitro and in vivo. PCR is used to confirm gene deletions in Trp53, Pten, and Rb1. Immunofluorescence (IF) staining of organoids demonstrates positive expression of urothelial lineage markers (CK5 and p63). The organoids are injected subcutaneously into host mice for tumor expansion and serial passages. The immunohistochemistry (IHC) of xenografts exhibits positive expression of CK7, CK5, and p63 and negative expression of CK8 and Uroplakin 3. In summary, adenovirus-mediated gene deletion from mouse urothelial cells engineered with loxP sites is an efficient method to rapidly test the tumorigenic potential of defined genetic alterations.

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

This protocol describes the process of the generation and characterization of mouse urothelial organoids harboring deletions in genes of interest. The methods include harvesting mouse urothelial cells, ex vivo transduction with adenovirus driving Cre expression with a CMV promoter, and in vitro as well as in vivo characterization.

离体 小鼠尿路上皮细胞腺病毒-Cre介导的基因缺失的类器官模型

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, senescence is a fundamentally different machinery restraining&#160;propagation of cancer cells. Decades of scientific studies have revealed the complex pathological effects of senescent cancer cells in tumors and microenvironments that modulate cancer cells and stromal cells. New evidence suggests that senescence is a potent prognostic factor during cancer treatment, and therefore rapid and accurate detection of senescent cells in cancer samples is essential. This paper presents a method to visualize and detect therapy-induced senescence (TIS) in cancer cells. Diffuse large B-cell lymphoma (DLBCL) cell lines were treated with mafosfamide (MAF) or daunorubicin (DN) and examined for the senescence marker, senescence-associated &#946;-galactosidase (SA-&#946;-gal), the DNA synthesis marker 5-ethynyl-2&#8242;-deoxyuridine (EdU), and the DNA damage marker gamma-H2AX (&#947;H2AX). Flow cytometer imaging can help generate high-resolution single-cell images in a short period of time to simultaneously visualize and quantify the three markers in cancer cells.

Here we present a flow cytometry-based method for visualization and quantification of multiple senescence-associated markers in single cells.

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...