Name: analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions
Uploaded: 2016-12-28T15:00:00.000+00:00
Duration: 10 min 40 s
Description: Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

This work was supported by the Natural Sciences and Engineering Council of Canada and the Ontario Ministry of Research and Innovation.

1.准备细胞培养<ol><li>购买人细胞的市售股票。 注:此协议利用HCT116结直肠癌和初级人肾小管上皮细胞（HRPTEC）。 </li><li>使对HCT116的培养500毫升培养基:Dulbecco改良的Eagle培养基（DMEM）中补充有7.5％胎牛血清（FBS）和1％青霉素/链霉素（P / S）/高葡萄糖培养基。 </li><li>作出HRPTEC的培养500毫升培养基:补充有5％FBS，1％的上皮细胞生长添加物上皮细胞培养基和1％P / S。 </li><li>制成500毫升之1×磷酸盐缓冲盐水（PBS）中的:140 mM氯化钠，3毫米氯化钾，10毫的 Na 2 HPO 4，15mM的KH 2 PO 4。调节pH至7.4，并通过高压灭菌121℃40分钟灭菌。 </li></ol> 2.细胞培养启动<ol><li>小心吸从80-90％汇合盘介质，而不会干扰Ť他的细胞。 </li><li>每培养皿等分试样3-5毫升1×PBS中，每个烧瓶轻轻摇动以覆盖培养皿的表面面积，并小心地吸出1×PBS中。重复第二1X PBS洗。 </li><li>等分试样的3ml的0.05％胰蛋白酶 - 乙二胺四乙酸（EDTA）到各培养皿并轻轻摇动以均匀涂层用胰蛋白酶-EDTA细胞。孵育在37℃下2-3分钟。 </li><li>转移分离的细胞入15ml离心管中并离心以4,000 xg离心90秒。 </li><li>在1毫升完全DMEM中悬浮细胞沉淀。 </li><li>算使用血球细胞。计算需要多少无热原和聚苯乙烯百毫米细胞培养皿以达到每平方厘米大约2,500-5,000细胞的初始接种密度。 </li><li>在步骤在37℃水浴中30-45分钟1.2或1.3制成预暖完整细胞培养基。 </li><li>净化培养基瓶和细胞小瓶，用70％的乙醇。从这点向前所有操作要无菌条件下进行。 </li><li>等分试样6毫升完全DMEM成使用在步骤2.6中提到的接种密度内冷冻细胞的整个体积需要作为100多MM细胞培养皿。 </li><li>传送在步骤2.6到每个100mm的培养皿计算细胞悬浮液的体积。手动摇动每个板到板的表面上均匀地分布的细胞。 </li><li>放置在孵化器中播种百毫米细胞培养皿于37℃在5％CO 2气氛中 。允许的下一步骤之前细胞孵育至少24小时。 </li></ol> 3.继代培养<ol><li>查看在显微镜下各细胞培养皿以确定细胞汇合的（％细胞覆盖培养皿的面积）的比例。返回细胞的孵化器和预温完全培养基和1x PBS。继续进行下一步骤，如果所述细胞是80-100％汇合。 </li><li>小心吸出用过的培养基，而不会干扰细胞在每个培养皿中。 </li><li>每培养皿等分试样3-5毫升1×PBS中，每个烧瓶轻轻摇动以覆盖培养皿的表面面积，并小心地吸出1×PBS中。重复第二1X PBS洗。 </li><li>等分试样3毫升0.05％胰蛋白酶-EDTA的到各培养皿并轻轻摇动以均匀涂层用胰蛋白酶-EDTA细胞。孵育在37℃下2-3分钟。 </li><li>在此孵化，分装在规定的10 ml完全培养基到尽可能多的培养皿获得每平方厘米2 2,500-5,000细胞的细胞密度。 </li><li>当细胞已经从板取下，迅速加入3毫升1×定义胰蛋白酶抑制剂和轻轻摇动培养皿1分钟，以确保所有的胰蛋白酶-EDTA的已中和。 </li><li>转移分离的细胞进入无菌15ml离心管中，放在一边。添加3毫升1×PBS中的菜，以收集任何残留的细胞，并传送到相同的15ml离心管中。 </li><li>通过离心150 XG颗粒细胞5分钟。 </li><li>吸出上清液，重悬细胞沉淀在3毫升完全培养基。 </li><li>算使用血球细胞和种子含有15 ml完全培养基，得到每平方厘米2,500-5,000细胞的细胞密度150毫米培养皿。使用两个150毫米每个帽结合试验。 注:通过液体介质氧扩散到细胞是依赖于量36。它建议保持实验之间保持一致的介质的体积是否使用在悬浮粘附细胞或细胞。媒体可以预调节（在工作站孵育如讨论讨论24小时），以避免在扩散这些变异。 </li><li>放置在孵化器中播种培养皿于37℃在5％CO 2气氛中 。允许在进行下一步骤之前，细胞孵育至少24小时。 </li></ol> 4. Physioxic曝光<ol><li>查看在显微镜下的细胞。对于BES吨的结果，确保细胞是一个24小时曝光70-80％汇合，50-60％汇合为一个48小时的曝光，而对于72小时曝光40-50％汇合。 </li><li>放置细胞进入所需时间（24，48，或72小时取决于实验）缺氧工作站。 </li><li>设置工作站到适当physioxia。 注:例如，3％O 2的设置将5％ 的 CO 2和92％N 2陪同。 注:如果动态范围内的氧气波动具体时间表，节目时间表所希望的过到仪器或手动调节在所需的时间间隔中的氧设置。 </li><li>湿度设定为60％。该工作站的气氛非常干燥尽管如此，和媒体将在很长的实验（&gt; 48小时）显着蒸发。保持在细胞培养烧瓶介质储备在工作站内（使介质与工作站中平衡），以补充介质在细胞培养DIS他是。 注意:任何解决方案，将与之前的裂解（例如PBS和胰蛋白酶-EDTA）的细胞相互作用应预调节24小时的缺氧工作站内使用前，使溶解氧与大气中的氧气36达到平衡。 </li><li>保持工作站，直到裂解细胞内。 </li></ol> 5.准备缓冲器，用于帽结合测定<ol><li>制备1×Tris-缓冲盐水（TBS）。 <ol><li>在800毫升的dh 2 O中，溶解8.76克NaCl和6.05克Tris碱。 </li><li>使溶液以使用1M的HCl的7.4的最终pH值。 </li><li>使终体积到使用DH 2 O 1升</li></ol></li><li>制备非变性裂解缓冲液。制备裂解缓冲帽结合测定当天。作出额外的裂解液洗空白琼脂糖珠和γ氨基苯基M 7 GTP琼脂糖C10联珠（步骤7.9和7.13）。 <ol><li> 7毫升的dh 2 O，加160微升5米NAC升，160微升的1M的Tris-HCl pH 7.4的40微升的200mM的NaF，40微升的1M MgCl 2的，和40微升1 1mM原钒酸钠。 </li><li>添加40微升的Igepal至7 ml溶液。切吸管尖，以帮助找回的Igepal，因为它是非常粘稠。 </li><li>吹打向上和向下，或涡流混合，7 ml溶液，直到所有的Igepal已经解散。 </li><li>升高溶液至8毫升的最终体积，并保持在冰上。 </li></ol></li><li>准备4X十二烷基硫酸钠（SDS）-PAGE样品缓冲液。 <ol><li>在一个烧杯中，将结合16毫升的1M的Tris-HCl pH 6.8的，12.64毫升甘油，和8毫升二硫苏糖醇（DTT）。 </li><li>添加3.2克SDS和0.16g溴酚蓝。允许粉末通过用磁力搅拌棒混合充分溶解。 </li><li>分装成的1.5 ml离心管中并储存在-20°C。 </li></ol></li></ol> 6.细胞裂解<ol><li>制备非变性裂解缓冲液，并保持在冰上日的日Ë帽结合试验。 </li><li>预暖1×PBS中和胰蛋白酶在37℃水浴中30分钟。 </li><li>正在执行等分试样980微升裂解缓冲液成每个帽结合测定1.5ml微量离心管中。 </li><li>添加4-（2-氨基乙基）苯磺酰氟盐酸盐的10微升和10微升100×蛋白酶抑制剂鸡尾酒的到980微升裂解缓冲液中。置于冰上。 </li><li>丢弃以150毫米的菜媒体在缺氧工作站内的废弃物容器中。 </li><li>清洗细胞3-4毫升温1X PBS，并放弃所有的液体。 </li><li> 1毫升温暖0.05％胰蛋白酶-EDTA添加到每个培养皿让坐在2分钟，或直至细胞不再附着到板上。 </li><li>使用移液管，一个板的细胞转移到1.5ml微量离心管中。根据需要，使细胞的每个板被转移到一个单独的1.5 ml离心管重复。 </li><li>离心1.5 ml离心管在6000 XG 90秒ŤØ颗粒细胞。 </li><li>吸用吸管胰蛋白酶，而不会干扰沉淀。如果颗粒被干扰，重新离心1.5 ml离心管在6000×g离心90秒。 </li><li>轻轻吹打200微升PBS的入管刚好沉淀上述要用温水1X PBS细胞。重新离心如果粒料被干扰。 </li><li>吸出PBS，而不会干扰沉淀。 </li><li>从1毫升吸管将500μl制备的裂解缓冲溶液到第一蜂窝沉淀。完全由上下吹打悬浮颗粒。 </li><li>结合重悬的细胞与所述第二细胞沉淀并通过上下抽吸悬浮所述第二沉淀。直到所有的粒料已经被组合和再悬浮每个样品重复此过程。 </li><li>结合裂解物与在1.5ml微量离心管中剩余的500μl的裂解缓冲液中。 </li><li>从缺氧工作站删除样本。 注:加入裂解缓冲液之后，将LL后续步骤如缺氧工作站被设定为37℃及以下的步骤将被执行的冷（4℃或冰上）在环境空气中进行。 </li><li>裂解用轻轻搅动在4℃下旋转样品1.5-2小时的细胞。 </li><li>离心裂解的细胞以12,000 xg离心15分钟，在4℃以除去细胞碎片。 </li><li>转移裂解物到新的1.5 ml离心管并弃沉淀。 </li><li>保留上清的一部分（5-10％），以用作将来Western印迹分析全细胞裂解物的输入控制。 </li></ol> 7.帽结合测定<ol><li>对于每个样品，转移空白琼脂糖珠控制浆料的50微升和50微升γ氨基苯基-间7 GTP琼脂糖C10联珠浆液以分离1.5ml微量管中。用剪刀取出吸移管的尖端，以便于珠浆的汇编。 </li><li>颗粒珠bý离心在500 xg离心浆液30秒。 </li><li>小心地取出上清，500微升TBS重悬珠。 </li><li>重复步骤7.2和7.3。沉淀小珠，在500×g离心30秒，并去除上清。 </li><li>从步骤6.19传送包含裂解物上清液到包含空白琼脂糖珠的1.5 ml离心管。 注:空白琼脂糖珠充当信息预先步骤，以从非特异性地与胎圈交互的裂解物除去蛋白质。 </li><li>孵育在4℃下10分钟，温和搅拌。 </li><li>通过在500 xg离心离心30秒沉淀空白琼脂糖珠。 </li><li>转移裂解物到含有γ氨基苯基-间7 GTP琼脂糖C10联珠的1.5 ml离心管。 </li><li>通过再悬浮于500μl裂解缓冲液的珠子，通过在500 xg离心离心30秒造粒的珠，并丢弃superna洗空白琼脂糖珠重要的。重复此四次总共五个洗涤。 </li><li>重悬在1×SDS-PAGE样品缓冲液空白琼脂糖珠，并在95℃下沸腾的珠90秒。储存在-20℃下以供将来Western印迹分析，观察感兴趣的蛋白质是否结合非特异性到胎圈。 </li><li>孵育轻轻摇动的γ氨基苯基-间7 GTP琼脂糖C10联珠1小时的溶胞产物在4℃下以捕获帽结合蛋白。 </li><li>通过在500 xg离心离心30秒沉淀γ氨基苯基-间7 GTP琼脂糖C10联的珠。弃去上清液。 </li><li>通过重复步骤7.9洗γ氨基苯基-间7 GTP琼脂糖C10联的珠。 </li><li>重悬在600微升裂解缓冲液的珠子。 </li><li>添加GTP到1mM的终浓度。 </li><li>孵育γ氨基苯基-间7 GTP琼脂糖C10联珠+ 1mM的GTP轻轻搅动在4℃下1小时。注:此将离解非特异性具有m 7 GTP相互作用的蛋白质（ 即，也与非甲基GTP交互）。 </li><li>通过在500 xg离心离心30秒沉淀γ氨基苯基-间7 GTP琼脂糖C10联的珠。 </li><li>将上清转移至新的1.5 ml离心管，加入200微升4×SDS-PAGE样品缓冲液（50mM的Tris-HCl，100mM的DTT，2％SDS，0.1％溴酚蓝和10％甘油）中的。储存在-20℃下对GTP对照样品供将来免疫印迹分析37。重复步骤7.9洗珠。 </li><li>重悬在1×SDS-PAGE样品缓冲液（50mM的Tris-HCl，100mM的DTT，2％SDS，0.1％溴酚蓝和10％甘油）中，并煮沸珠粒在95℃下90秒。储存在-20℃第m 7 GTP结合的级分以供将来免疫印迹分析37。 </li></ol>

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The authors declare that they have no competing financial interests.

在暴露于生理氧条件的人细胞帽结合蛋白质的分析可允许的新颖氧 - 调节翻译起始因子的鉴定。这些因素为mRNA或其它帽相关蛋白的5&#39;帽的亲和性可以通过其关联的强度被测量到m 7 GTP联的琼脂糖珠。这种技术的一个需要注意的是它测量蛋白裂解后的帽结合的潜力，但它是该保持蛋白质 - 蛋白质相互作用和翻译后后修饰非变性条件下进行。几个蛋白 - 蛋白相互作用介导的eIF4E家族的5&#39;帽?...

平移控制正在成为基因表达的转录调节同样重要的一步，尤其是在细胞应激1期。翻译调控的一个重点是在（M 7 GTP）5&#39;的mRNA 2帽引发的限速步骤，其中的蛋白质合成的第一个步骤涉及真核细胞起始因子4E（eIF4E的）的结合到7-甲基。 eIF4E的是一个名为eIF4F三聚体复杂，包括eIF4A，一个RNA解旋酶和eIF4G，对其他翻译因子和核糖体40S 3招聘要求支架蛋白的一部分。在正常生理条件下，绝大多数的mRNA经由帽依赖性机制翻译，但在细胞应激期间人类mRNA的约10％含有5&#39;非编码区，可能允许不依赖帽的翻译intiation 1,4。帽依赖性翻译在历史上同义词OU中与eIF4F，但是，eIF4F的特定压力变化已经成为一个热门话题5-8。 各种细胞应激引起雷帕霉素通过复杂1（mTORC1的）的哺乳动物目标受到抑制eIF4E的活动。这种激酶变得在压力下，这导致在其目标之一的增加的活性受损，所述4E结合蛋白（4E-BP）。非磷酸化4E-BP结合eIF4E与阻断其与eIF4G引起帽依赖性翻译9,10的压制交往的能力。有趣的是，一个名为eIF4E2（或4EHP）的eIF4E的同源物具有4E-BP 11低得多的亲和力，或许允许它逃避压力介导的镇压。事实上，最初表征为翻译阻遏由于其缺乏与eIF4G 12相互作用，eIF4E2发起数百包含在其3&#39;端非编码区的RNA缺氧反应元件的mRNA的低氧应激6,13-在翻译。这种激活我s至与eIF4G3，RNA结合蛋白基序4，和低氧诱导因子（HIF）2α构成低氧eIF4F复杂，或eIF4Fħ6,13-相互作用来实现的。如正常条件下的阻遏，eIF4E2结合与GIGYF2和ZNF598 14。这些复合物，在部分地通过琼脂糖联M 7 GTP的亲和树脂识别。这种传统方法15在翻译领域的标准，是最好的，最常用的技术，在下拉隔离帽结合复合物和体外结合试验16-19。作为帽依赖性翻译机器正在成为灵活性和适应性与间变化部分6-8,13，这种方法是一个强大的工具，迅速查明参与应激反应小说帽结合蛋白。此外，eIF4F的变化可能有广泛影响几个真核模型系统出现使用eIF4E2同源的应激反应等如拟南芥 20， 裂殖酵母 21， 果蝇 22和线虫 23。 有证据表明，在eIF4F变化可能不严格限于强调的条件，但参与正常生理学24。氧气供给到组织中（在毛细管两端）或组织中（通过微电极测定）从2-6％在脑中25而变化，在肺26 3-12％，在肠道27，在4％3.5-6％肝脏28，在肾脏29 7-12％，在肌肉30 4％，在骨髓中31 6-7％。细胞线粒体含有比1.3％的氧气少32。这些值比周围的空气，其中细胞常规培养更接近缺氧。这表明什么以前被认为是特定缺氧细胞过程可以是在生理环境相关。有趣的是，eIF4F和eIF4F ^ h </suP&gt;积极参与不同池或mRNA的类的翻译起始于暴露于生理氧或"physioxia"24几个不同的人细胞系。低氧也推动适当的胎儿发育33和电池通常具有较高的增殖率，寿命长，少的DNA损伤和physioxia 34较少的一般应激反应。因此，eIF4F H是可能在生理条件下选择的基因的表达的关键因素。 这里，我们提供了一个协议，以在固定的生理氧条件下或在这是可能的更具代表性的组织微环境的动态波动范围培养的细胞。这种方法的一个优点是，细胞在缺氧工作站内裂解。它是不是经常不清楚如何从缺氧细胞培养细胞裂解转变在其他协议执行。细胞常常首先从一个小缺氧孵化器中删除是前的裂解，但是这种暴露于氧气可能影响生化途径以氧气的细胞反应是快速（一个或两个分）35。某些帽结合蛋白需要用第二碱相互作用或能水解第m 7 GTP，因此一些帽交互件可在纯化过程被遗漏。琼脂糖联以酶促抗帽类似物可以在该协议被取代。探索通过这里描述的方法的活性和eIF4F H和eIF4F的其它变型的组合物将在该细胞中的生理条件或胁迫应答利用复杂的基因表达的机械线索。

<table><tbody><tr><td>&amp;gamma;-aminophenyl-m&lt;sup&gt;7&lt;/sup&gt;GTP agarose C&lt;sub&gt;10&lt;/sub&gt;-linked beads</td><td>Jena Bioscience</td><td>AC-1555</td><td>Agarose-linked m&lt;sup&gt;7&lt;/sup&gt;GTP</td></tr><tr><td>100 mm culture dish</td><td>Corning</td><td>877222</td><td>10-cm culture dish</td></tr><tr><td>150 mm culture dish</td><td>Thermofisher</td><td>130183</td><td>15 cm culture dish</td></tr><tr><td>AEBSF Hydrochloride</td><td>ACROS Organics</td><td>A0356829</td><td>AEBSF</td></tr><tr><td>Agarose Beads</td><td>Jena Bioscience&amp;nbsp;</td><td>AC-0015</td><td>Agarose bead control</td></tr><tr><td>Bromophenol Blue</td><td>Fisher</td><td>BP112-25</td><td>Component of SDS-PAGE loading buffer</td></tr><tr><td>1.5 ml Centrifuge Tubes</td><td>FroggaBio</td><td>1210-00S</td><td>Used to centrifuge small volumes</td></tr><tr><td>15 ml Conical Centrifuge Tubes</td><td>Fisher</td><td>1495970C</td><td>Used in culturing primary cells</td></tr><tr><td>Defined trypsin inhibitor</td><td>Fisher</td><td>R007100</td><td>DTI</td></tr><tr><td>Dithiothreitol</td><td>Fisher</td><td>BP172-25</td><td>DTT</td></tr><tr><td>Epithelial cell medium (complete kit)</td><td>ScienCell</td><td>4101</td><td>Includes serum and growth factor supplements)</td></tr><tr><td>Glycerol</td><td>Fisher</td><td>BP229-1</td><td>Component of SDS-PAGE loading buffer</td></tr><tr><td>100 mM Guanosine 5&#039;-triphosphate, 1 ml</td><td>Jena Bioscience</td><td>272076-0251M</td><td>GTP</td></tr><tr><td>HCT116 colorectal carcinoma</td><td>ATCC</td><td>CCL-247</td><td>Human cancer cell line</td></tr><tr><td>Human renal proximal tubular epithelial cells</td><td>ATCC</td><td>PCS-400-010</td><td>HRPTEC</td></tr><tr><td>Hyclone DMEM/High Glucose</td><td>GE Life Sciences</td><td>SH30022.01</td><td>Standard media for human cell culture</td></tr><tr><td>Hyclone Penicillin-Streptomycin solution</td><td>GE Life Sciences</td><td>SV30010</td><td>Antibiotic component of DMEM</td></tr><tr><td>H35 HypOxystation</td><td>Hypoxygen</td><td>N/A</td><td>Hypoxia workstation</td></tr><tr><td>Igepal CA-630</td><td>MP Biomedicals</td><td>2198596</td><td>Detergent component of lysis buffer</td></tr><tr><td>Monopotassium phosphate</td><td>Fisher</td><td>P288-500</td><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Potassium chloride</td><td>Fisher</td><td>P217-500</td><td>KCl</td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>M33-500</td><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td></tr><tr><td>Sodium chloride</td><td>Fisher</td><td>BP358-10</td><td>NaCl</td></tr><tr><td>Sodium fluoride</td><td>Fisher</td><td>5299-100</td><td>NaF (phosphatase inhibitor component of lysis buffer)</td></tr><tr><td>Disodium phosphate</td><td>Fisher</td><td>5369-500</td><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Premium Grade Fetal Bovine Serum</td><td>Seradigm</td><td>1500-500</td><td>FBS</td></tr><tr><td>Protease Inhibitor Cocktail (100x)</td><td>Cell Signalling</td><td>58715</td><td>Component of lysis buffer</td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Fisher</td><td>BP166-100</td><td>SDS</td></tr><tr><td>Sodium Orthovanadate</td><td>Sigma</td><td>56508</td><td>Na&lt;sub&gt;3&lt;/sub&gt;VO&lt;sub&gt;4&lt;/sub&gt;</td></tr><tr><td>Tris Base</td><td>Fisher</td><td>BP152-5</td><td>Component of buffers</td></tr><tr><td>0.05% Trypsin-EDTA (1x)</td><td>Life Technologies</td><td>2500-067</td><td>Trypsin used to detach adherent cells</td></tr></tbody></table>

analysis of cap-binding proteins in human cells exposed to physiological oxygen conditions

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex is by far the most common pathway to initiate protein synthesis in eukaryotic cells, but stress-specific variations of this complex are now emerging. Purifying cap-binding proteins with an affinity resin composed of Agarose-linked m7GTP (a 5' mRNA cap analog) is a useful tool to identify factors involved in the regulation of translation initiation. Hypoxia (low oxygen) is a cellular stress encountered during fetal development and tumor progression, and is highly dependent on translation regulation. Furthermore, it was recently reported that human adult organs have a lower oxygen content (physioxia 1-9% oxygen) that is closer to hypoxia than the ambient air where cells are routinely cultured. With the ongoing characterization of a hypoxic eIF4F complex (eIF4FH), there is increasing interest in understanding oxygen-dependent translation initiation through the 5' mRNA cap. We have recently developed a human cell culture method to analyze cap-binding proteins that are regulated by oxygen availability. This protocol emphasizes that cell culture and lysis be performed in a hypoxia workstation to eliminate exposure to oxygen. Cells must be incubated for at least 24 hr for the liquid media to equilibrate with the atmosphere within the workstation. To avoid this limitation, pre-conditioned media (de-oxygenated) can be added to cells if shorter time points are required. Certain cap-binding proteins require interactions with a second base or can hydrolyze the m7GTP, therefore some cap interactors may be missed in the purification process. Agarose-linked to enzymatically resistant cap analogs may be substituted in this protocol. This method allows the user to identify novel oxygen-regulated translation factors involved in cap-dependent translation.

在一个m 7 GTP亲和柱的响应eIF4E与eIF4E2氧帽结合力分析 图1和2表示响应于氧的波动在两个人细胞系中两个主要的帽结合蛋白的典型M 7 GTP的亲和纯化的蛋白质印迹:原代人肾小管上皮F中igure 1和大肠癌细胞（HRPTEC）（ HCT116）在图2中。细胞维持在所指示的氧气供应24小?...

Watch this Scientific Journal Video about Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions at JoVE.com

Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

Translational control is a focal point of gene regulation, especially during periods of cellular stress. Cap-dependent translation via the eIF4F complex ...

analysis-cap-binding-proteins-human-cells-exposed-to-physiological

Research

JoVE Journal

Genetics

7.7K 次观看.  University of Guelph. Here, we present human cell culture protocols to analyze translation initiation factors that bind the 5' cap of mRNA during physiological oxygen conditions. This method utilizes an Agarose-linked m7GTP cap analog and is suitable to investigate cap-binding factors and their interacting partners.

视频: Analysis of Cap-binding Proteins in Human Cells Exposed to Physiological Oxygen Conditions

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the gene is regulated by the particular timing of one of many stimuli, signaling pathways or secondary effects. In order to capture the entire gene expression response to hypoxia in the yeast S. cerevisiae, RNA-seq analysis was used to monitor the mRNA levels of all genes at specific times after exposure to hypoxia. Hypoxia was established by growing cells in ~100% N2 gas. Importantly, unlike other hypoxic studies, ergosterol and unsaturated fatty acids were not added to the media because these metabolites affect gene expression. Time points were chosen in the range of 0 - 4 h after hypoxia because that period captures the major changes in gene expression. At each time point, mid-log hypoxic cells were quickly filtered and frozen, limiting exposure to O2 and concomitant changes in gene expression. Total RNA was extracted from cells and used to enrich for mRNA, which was then converted to cDNA. From this cDNA, multiplex libraries were created and eight or more samples were sequenced in one lane of a next-generation sequencer. A post-sequencing pipeline is described, which includes quality base trimming, read mapping and determining the number of reads per gene. DESeq2 within the R statistical environment was used to identify genes that change significantly at any one of the hypoxic time points. Analysis of three biological replicates revealed high reproducibility, genes of differing kinetics and a large number of expected O2-regulated genes. These methods can be used to study how the cells of various organisms respond to hypoxia over time and adapted to study gene expression during other cellular responses.

Measuring mRNA Levels Over Time During the Yeast S. cerevisiae Hypoxic Response

Here, we present a protocol using RNA-seq to monitor mRNA levels over time during the hypoxic response of S. cerevisiae cells. This method can be adapted to analyze gene expression during any cellular response.

在酵母S.酵母缺氧反应期间, 测量一段时间内的 mRNA 水平

Complex changes in gene expression typically mediate a large portion of a cellular response. Each gene may change expression with unique kinetics as the ...

科研

遗传学

Tracking Hypoxic Signaling within Encapsulated Cell Aggregates

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal hyperglycemia. As an alternative treatment option to exogenous insulin injection, transplantation of functional pancreatic tissue has been explored1,2. This approach offers the promise of a more natural, long-term restoration of normoglycemia. Protection of the donor tissue from the host's immune system is required to prevent rejection and encapsulation is a method used to help achieve this aim.
Biologically-derived materials, such as alginate3 and agarose4, have been the traditional choice for capsule construction but may induce inflammation or fibrotic overgrowth5 which can impede nutrient and oxygen transport. Alternatively, synthetic poly(ethylene glycol) (PEG)-based hydrogels are non-degrading, easily functionalized, available at high purity, have controllable pore size, and are extremely biocompatible,6,7,8. As an additional benefit, PEG hydrogels may be formed rapidly in a simple photo-crosslinking reaction that does not require application of non-physiological temperatures6,7. Such a procedure is described here. In the crosslinking reaction, UV degradation of the photoinitiator, 1-[4-(2-Hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propane-1-one (Irgacure 2959), produces free radicals which attack the vinyl carbon-carbon double bonds of dimethacrylated PEG (PEGDM) inducing crosslinking at the chain ends. Crosslinking can be achieved within 10 minutes. PEG hydrogels constructed in such a manner have been shown to favorably support cells7,9, and the low photoinitiator concentration and brief exposure to UV irradiation is not detrimental to viability and function of the encapsulated tissue10. While we methacrylate our PEG with the method described below, PEGDM can also be directly purchased from vendors such as Sigma.
An inherent consequence of encapsulation is isolation of the cells from a vascular network. Supply of nutrients, notably oxygen, is therefore reduced and limited by diffusion. This reduced oxygen availability may especially impact &beta;-cells whose insulin secretory function is highly dependent on oxygen11-13. Capsule composition and geometry will also impact diffusion rates and lengths for oxygen. Therefore, we also describe a technique for identifying hypoxic cells within our PEG capsules. Infection of the cells with a recombinant adenovirus allows for a fluorescent signal to be produced when intracellular hypoxia-inducible factor (HIF) pathways are activated14. As HIFs are the primary regulators of the transcriptional response to hypoxia, they represent an ideal target marker for detection of hypoxic signaling15. This approach allows for easy and rapid detection of hypoxic cells. Briefly, the adenovirus has the sequence for a red fluorescent protein (Ds Red DR from Clontech) under the control of a hypoxia-responsive element (HRE) trimer. Stabilization of HIF-1 by low oxygen conditions will drive transcription of the fluorescent protein (Figure 1). Additional details on the construction of this virus have been published previously15. The virus is stored in 10% glycerol at -80&deg; C as many 150 &mu;L aliquots in 1.5 mL centrifuge tubes at a concentration of 3.4 x 1010 pfu/mL. 
Previous studies in our lab have shown that MIN6 cells encapsulated as aggregates maintain their viability throughout 4 weeks of culture in 20% oxygen. MIN6 aggregates cultured at 2 or 1% oxygen showed both signs of necrotic cells (still about 85-90% viable) by staining with ethidium bromide as well as morphological changes relative to cells in 20% oxygen. The smooth spherical shape of the aggregates displayed at 20% was lost and aggregates appeared more like disorganized groups of cells. While the low oxygen stress does not cause a pronounced drop in viability, it is clearly impacting MIN6 aggregation and function as measured by glucose-stimulated insulin secretion15. Western blot analysis of encapsulated cells in 20% and 1% oxygen also showed a significant increase in HIF-1&alpha; for cells cultured in the low oxygen conditions which correlates with the expression of the DsRed DR protein.

A method for photo-encapsulation of cells in a crosslinked PEG hydrogel is described. Hypoxic signaling within encapsulated murine insulinoma (MIN6) aggregates is tracked using a fluorescent marker system. This system allows serial examination of cells within a hydrogel scaffold and correlation of hypoxic signaling with changes in cell phenotype.

在封装的细胞聚集跟踪缺氧信号

In Diabetes mellitus type 1, autoimmune destruction of the pancreatic &beta;-cells results in loss of insulin production and potentially lethal ...

生物工程

Proteomic Analysis of Human Macrophage Polarization Under a Low Oxygen Environment

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. The various functions of these cells are related to their activation states, which is also called polarization. The precise molecular description of these various polarizations is a priority in the field of macrophage biology. It is currently acknowledged that a multidimensional approach is necessary to describe how polarization is controlled by environmental signals. In this report, we describe a protocol designed to obtain the proteomic signature of various polarizations in human macrophages. This protocol is based on a label-free quantification of macrophage protein expression obtained from in-gel fractionated and Lys C/trypsin-digested cellular lysis content. We also provide a protocol based on in-solution digestion and isoelectric focusing fractionation to use as an alternative. Because oxygen concentration is a relevant environmental parameter in tissues, we use this protocol to explore how atmospheric composition or a low oxygen environment affects the classification of macrophage polarization.

We present a protocol to obtain proteomic signatures of human macrophages and apply this to determination of the impact of a low oxygen environment on macrophage polarization.

低氧环境下人巨噬细胞极化的蛋白质组学分析

Macrophages are innate immune cells involved in a number of physiological functions ranging from responses to infectious pathogens to tissue homeostasis. ...

生物化学

稳定同位素追踪和LC-MS分析以测量DHODH和SDH活性

Oxygen-Independent Assays to Measure Mitochondrial Function in Mammals

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in mammalian cells. As oxygen (O2) is the most ubiquitous terminal electron acceptor for the mammalian ETC, the O2 consumption rate is frequently used as a proxy for mitochondrial function. However, emerging research demonstrates that this parameter is not always indicative of mitochondrial function, as fumarate can be employed as an alternative electron acceptor to sustain mitochondrial functions in hypoxia. This article compiles a series of protocols that allow researchers to measure mitochondrial function independently of the O2 consumption rate. These assays are particularly useful when studying mitochondrial function in hypoxic environments. Specifically, we describe methods to measure mitochondrial ATP production, de novo pyrimidine biosynthesis, NADH oxidation by complex I, and superoxide production. In combination with classical respirometry experiments, these orthogonal and economical assays will provide researchers with a more comprehensive assessment of mitochondrial function in their system of interest.

作者聚焦：测量哺乳动物线粒体功能的氧非依赖型检测  

Here, we present a compilation of assays to directly measure mitochondrial function in mammalian cells independently of their ability to consume molecular oxygen.

测量哺乳动物线粒体功能的氧非依赖性测定

The flow of electrons in the mitochondrial electron transport chain (ETC) supports multifaceted biosynthetic, bioenergetic, and signaling functions in ...

Respirometric Oxidative Phosphorylation Assessment in Saponin-permeabilized Cardiac Fibers

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, oxidative stress, apoptosis, aging, mitochondrial encephalomyopathies and drug toxicity. Given this, technologies to measure cardiac mitochondrial function are in demand. One technique that employs an integrative approach to measure mitochondrial function is respirometric oxidative phosphorylation (OXPHOS) analysis.
The principle of respirometric OXPHOS assessment is centered around measuring oxygen concentration utilizing a Clark electrode. As the permeabilized fiber bundle consumes oxygen, oxygen concentration in the closed chamber declines. Using selected substrate-inhibitor-uncoupler titration protocols, electrons are provided to specific sites of the electron transport chain, allowing evaluation of mitochondrial function. Prior to respirometric analysis of mitochondrial function, mechanical and chemical preparatory techniques are utilized to permeabilize the sarcolemma of muscle fibers. Chemical permeabilization employs saponin to selectively perforate the cell membrane while maintaining cellular architecture.
This paper thoroughly describes the steps involved in preparing saponin-skinned cardiac fibers for oxygen consumption measurements to evaluate mitochondrial OXPHOS. Additionally, troubleshooting advice as well as specific substrates, inhibitors and uncouplers that may be used to determine mitochondria function at specific sites of the electron transport chain are provided. Importantly, the described protocol may be easily applied to cardiac and skeletal tissue of various animal models and human samples.

Saponin-permeabilized fiber preparation in conjunction with respirometric oxidative phosphorylation analysis provides integrative assessment of mitochondrial function. Mitochondrial respiration in physiological and pathological states can reflect various regulatory influences including mitochondrial interactions, morphology and biochemistry.

Respirometric皂甙通透心脏纤维的氧化磷酸化的评估

Investigation of mitochondrial function represents an important parameter of cardiac physiology as mitochondria are involved in energy metabolism, ...

生物学

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

糖原生化滴定<em&gt;在体外</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

全球合成的RNA在单细胞水平以下的低氧分析

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

暴露在生理条件下的氧气在人体细胞中帽结合蛋白质分析

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