这项工作得到了国家卫生和医学研究理事会 (澳洲) 给 H-爵士的赠款的支持。TMBW 是澳大利亚研究生奖的接受者澳大利亚政府和 Ann 亨德森的顶部奖学金从澳大利亚扶轮健康与扶轮 Templestowe 和用餐的治疗。

所有动物实验都是由墨尔本大学动物伦理委员会批准的。
注: 腺病毒载体的序列, 构造和生成协议 pad-巨细胞病毒-Td-汤姆, pad cmv-GFP和 pad-CAGA12-卢克/Td-汤姆以前被描述了11,12, 13。所有的向量都是商用的。
1. 使用50% 组织培养感染剂量的病毒效价测定 (TCID50)
<ol><li>准备 1 x 106 HEK293A 细胞在10毫升的 DMEM + 10% 的血清介质。</li>
	<li>种子100µL 细胞悬浮在一个96井平底细胞培养板的每一个井。</li>
	<li>在完全培养基中制备1毫升1:100 稀释的病毒储存。将稀释病毒的11µL 添加到1列的每个井中。</li>
	<li>通过将稀释病毒的11µL 与100µL 细胞悬浮液混合, 在96井板中直接进行10倍稀释, 直到达到 1 x1012的最终稀释。虽然细胞在这个阶段是不黏附的, 在井之间转移的细胞的数量保持恒定。</li>
	<li>吸管11µL 每稀释入每列从最高的稀释 104开始。用每个稀释液取代吸管尖, 以限制病毒微粒的交叉。</li>
	<li>将11µL 无病毒的完整介质添加到12列作为负控制。</li>
	<li>孵育板材为 7-10 天在37°c 与 10% CO2。</li>
	<li>使用显微镜, 在每列的水井数中显示病变或细胞毒性效应的迹象。如果有任何感染迹象, 请考虑良好的阳性反应。</li>
	<li>用芦苇和明奇 (1938) 统计方法14计算每个稀释的正井的分数。</li>
	<li>计算比例距离 (PD) 使用: (50)/(a B), 其中 A 是百分比响应以上或等于50% 和 B 是百分比响应低于50%。</li>
	<li>计算 TCID50使用:10x PD, 其中 x 是稀释给予响应大于50%。</li>
	<li>使用以下方法计算病毒滴度: 0.69/(0.011 x TCID50) PFU/毫升</li>
</ol>2. 腺病毒的语言测定
<ol><li>种子 1.0-1.5 x 105 MDA-MB-231 细胞与 500 DMEM 培养基的 ul 在5% 个井板 (12 个细胞融合, 35% 个井一共) 的每井含6个血清。</li>
	<li>使用以下公式计算0、250、500、2500和5000语言所需的Ad 巨细胞病毒--Td-Tom的数量: 教学语言 = (容积 [ul] x PFU/ul)/细胞数。对于Ad 巨细胞病毒-Td-汤姆以 1 x 1010 (PFU/毫升) 的效价, 所需的感染量 mois 》杂志分别为0、3.75、7.5、37.5 和 75 ul。</li>
	<li>在2.2 节中, 通过直接将病毒吹打到水井中, 通过彻底混合, 感染经计算的Ad-CMV-Td-汤姆的细胞。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>从水井中取出介质, 并立即用 500 ul 10% 福尔马林的电池固定细胞, 5 分钟。</li>
	<li>用 500 ul 的 PBS 从水井中吸出福尔马林, 一次洗井。</li>
	<li>用 500 ul 赫斯特溶液染色细胞核, 5 分钟。</li>
	<li>取出赫斯特溶液, 用 PBS 500 ul 3 次冲洗水井。</li>
	<li>在荧光显微镜上使用200X 放大图像的 Td-番茄蛋白和细胞核信号。激发 Td-番茄蛋白在554毫微米和赫斯特染料在352毫微米。</li>
	<li>使用 ImageJ 分析图像, 确定100% 感染 Td-番茄15所需的工作语言。</li>
</ol>3. 活单细胞中的双腺病毒转导
<ol><li>种子 1.0-1.5 x 105 MDA-MB-231 细胞与 500 DMEM 培养基的 ul 在5% 个井板 (12 个细胞融合, 35% 个井一共) 的每井含2个血清。</li>
	<li>感染细胞与 75 ul 的ad CMV-GFP (效价 = 5 x 109 PFU/毫升) 和 7.5 ul 的ad CAGA12-Td-汤姆(效价 = 5 x 1010 PFU/毫升) 获得2500语言, 可以达到100% 感染阳性。</li>
	<li>治疗细胞与或没有 0.25 ul 的 TGF β每井 (10 毫克/毫升的库存, 5 ng/ul 作为最终浓度) 后立即腺病毒感染。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>图像活细胞与相对比荧光显微镜。激发 GFP 在470毫微米和 Td-蕃茄蛋白质在554毫微米。</li>
	<li>固定细胞和染色细胞核, 如步骤 1.6-1.9 所述。</li>
	<li>用荧光显微镜对 Td-番茄蛋白和赫斯特染色进行图像处理。激发赫斯特染料在352毫微米。</li>
	<li>使用 ImageJ, 计算 GFP 和 Td-番茄阳性细胞的百分比15。</li>
</ol>4. 活体单细胞信号在创面愈合试验中的作用
<ol><li>种子 4-5 x 105 MDA-MB-231 细胞与1毫升的 DMEM 培养基包含5% 个血清在6个井板 (50% 个细胞融合, 2 井总共) 的每井。</li>
	<li>感染细胞与 22.5 ul 的Ad CAGA12-Td-汤姆(效价 = 5 x 1010 PFU/毫升, 语言 = 2500)。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>用 P200 吸管尖端在每个井的细胞层上划两条交叉线。</li>
	<li>从水井中取出旧介质, 并用2毫升新鲜5% 的 TGF, 用或不含 0.5 ul 的每口井 (10 毫克/毫升的库存, 5 ng/ul 作为最终浓度)。</li>
	<li>将板材放在孵化器的37°c 与 10% CO2 5 分钟, 并采取在相对比显微镜上使用100X 放大的选择伤口区域的图像。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>在同一个区域拍摄伤口闭合图像。</li>
	<li>修复细胞, 染色细胞核, 如步骤 1.6-1.9 所述, 并可视化的 Td-番茄信号在伤口区域和非伤口区域使用200X 放大的荧光显微镜。</li>
	<li>使用 ImageJ 软件15, 量化伤口和非创面的 Td-番茄阳性细胞的百分比。</li>
</ol>5.体外荧光素酶测定
<ol><li>在1毫升的 DMEM 培养基中制备 3-5 x 104 MDA-MB-231 细胞悬浮液, 含5% 个血清。</li>
	<li>感染细胞悬浮与 2.5 ul 的Ad CAGA12-卢克(效价 = 5 x 1010 PFU/毫升, 语言 = 2500)。</li>
	<li>将受感染的细胞种子放入96井板, 在 3000 cells/100 µL/井。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>从水井中取出旧介质, 并用100µL 新鲜5% 的 TGF, 用或不含 0.1 ul (含1毫克/毫升的库存, 1 ng/ul 为最终浓度), 并在存在或没有 0.17 ul 的 TGF β抑制剂每井 (7 毫克/毫升的库存, 12 ng/ul 为最终浓度) (一种新的 TGF β配体诱捕蛋白, 陈等, 未发表的工作)。</li>
	<li>安置板材在孵化器在37°c 与 10% CO2为 24 h。</li>
	<li>在双蒸馏水中解冻荧光素酶检测系统基质, 并制备1x 细胞培养裂解试剂 (DDW)。</li>
	<li>从96口井和溶解井中取出介质, 用 50 ul 1x 细胞培养裂解缓冲冰, 使用一台带有温和搅拌的眼眶, 30 分钟。</li>
	<li>将 30 ul 的裂解液从每一个井转移到一个不透明的荧光素片中, 让盘子温暖室温。</li>
	<li>在紫外线软件上, 创建一个新的协议。选择1注射器的测量前后注射。在测量和积分时间的延迟前, 将测量值设置为1秒。在注射测量后, 将注入量设置为40µL, 在注射后1秒和 5 s积分时间内进行延迟测量。通过选择"确定"确认设置。</li>
	<li>将喷油器管放入 DDW 中, 然后单击最佳设置下的喷油器 1按钮, 以在使用前清洗喷油器管。下一步从 DDW 中取出喷油器管, 然后在空气中放置两个进一步的引射器 1, 以冲洗管子中的所有溶液。将喷油器管放入萤火虫基体中, 再用两次质数准备基体。</li>
	<li>将不透明的荧光素酶板与样品放到紫外线中, 然后选择软件上的选项。突出显示感兴趣的水井, 然后单击 "应用"。按开始测量荧光素酶信号。</li>
	<li>测量完成后, 用清水除去并冲洗盘子。将管子插入空气中, 然后将其注入1回萤火虫衬底管, 将剩余的基板恢复到喷油器管中。将喷油器管放入 DDW, 再执行两个素数, 以清洁任何剩余基体的喷油器管。</li>
</ol>6. 活体信号成像的细胞制备
<ol><li>48小时前肿瘤植入, 种子 2 3x106 MDA-MB-231 细胞成175厘米2瓶 (总共10瓶)。培养细胞在20毫升的 DMEM 培养基中含有5% 的血清在37摄氏度, 10% CO2。</li>
	<li>24小时后, 添加 300 ul 的Ad CAGA12-卢克(效价 = 5 x 1010 PFU/毫升, 语言 = 2500) 到每个烧瓶。保持细胞在文化中的24小时。</li>
	<li>在植入日, 取出旧的培养基, 用20毫升的 PBS, 7.4 的 pH 值清洗腺病毒感染的细胞。在烧瓶中加入2毫升0.05% 胰蛋白酶-EDTA, 然后稍微摇动烧瓶, 让胰蛋白酶覆盖所有的烧瓶表面。放置烧瓶到孵化器37°c, 10% CO2为3分钟。</li>
	<li>将8毫升的含 DMEM 培养基加入烧瓶中, 使胰蛋白酶淬火。将所有细胞悬浮液转移到玻璃试剂瓶中。</li>
	<li>使用 hemocytometer 计数细胞数, 并在两个50ml 离心管中传输 4.8 x 107细胞。</li>
	<li>离心细胞在 400 x g 5 分钟的 RT, 以去除胰蛋白酶和并用重悬细胞在720毫升的新鲜的 DMEM 培养基。</li>
	<li>将细胞悬浮液转移到不育的5毫升管中, 加入 160 ul 胶 (10% 总胶悬浮液)。</li>
	<li>把细胞放在冰上直到植入。</li>
</ol>7. 原位植入术
<ol><li>重12只免疫缺陷小鼠, 随机将小鼠分配到控制和治疗组 (6 只小鼠/组)。</li>
	<li>在无菌的生物箱中进行植入, 以保持不育的环境。麻醉鼠标使用 2.5-4.5% 异氟醚, 氧流速为1升/分. 将鼠标放在热垫上, 并将一滴润滑眼膏放在眼睛上, 以避免角膜损伤。将鼠标固定在仰卧位的热垫上, 同时通过将鼻口放入与异氟醚相连的鼻锥中保持麻醉。</li>
	<li>通过对脚趾捏缺乏反应来确认麻醉的成功。减少异氟醚的维持剂量为 1.5-2.5% 与1升/分钟氧气提醒植入。</li>
	<li>用棉签蘸上80% 乙醇, 从两侧的第四奶嘴中清除皮肤。</li>
	<li>通过触诊找到4乳房脂肪垫, 挤压脂肪垫进一步暴露组织。</li>
	<li>通过吹打上下混合, 使细胞悬浮良好。轻轻地将50µL 的细胞混合物吸入27G 胰岛素注射器中, 并注入鼠标两侧的乳房脂肪垫。</li>
	<li>确认成功注射的脂肪垫肿胀的感觉。轻轻松开脂肪垫, 把鼠标放回笼子里。</li>
	<li>把笼子放在热垫上至少30分钟, 这样老鼠才能获得知觉, 然后把笼子放回拘留室3天。</li>
</ol>8. IVIS 成像
<ol><li>打开活生生的图像软件。</li>
	<li>通过单击 "控制面板" 上的 "初始化 IVIS 系统" 按钮初始化 IVIS 系统。等到温度标志变绿。</li>
	<li>在 "控制面板" 中选择发光成像模式。</li>
	<li>将异氟醚麻醉转向腔室和 IVIS 系统。</li>
	<li>用 2.5-4.5% 异氟醚与1升/分氧麻醉小鼠。一旦麻醉, 将异氟醚设置为 1.5-2.5% 以维护。用腹腔 (ip) 注射剂将 d-荧光素的150毫克/千克体重注入小鼠体内。</li>
	<li>立即将小鼠转移到 IVIS 系统中, 把它们放到温度控制的成像平台上, 把鼻子放到鼻锥中。</li>
	<li>单击 "控制面板" 上的 "序列设置" 按钮, 然后选择 "介质 Binning"。</li>
	<li>设置成像曝光时间如下: 十年代、二十年代、三十年代、六十年代和 120s. 在荧光素注射后开始成像大约3分钟, 并继续图像直到信号开始下降 (通常这将需要大约20分钟)。</li>
	<li>从 IVIS 中去除小鼠, 同时保持异氟醚麻醉通过鼻锥和治疗小鼠与50µL PBS 或50µL TGF β信号抑制剂 (50 微克/肿瘤) 通过肿瘤内注射。</li>
	<li>把老鼠放回笼子里, 放在热垫上至少30分钟。然后把笼子放回拘留室。</li>
	<li>重复 IVIS 成像程序, 如上文所述治疗后的前一天。</li>
</ol>9. 数据分析
<ol><li>在活体图像软件中打开保存的文件。</li>
	<li>使用视图查找图像信息 |图像信息。</li>
	<li>选择在同一时间点拍摄的照片, 同时曝光时间相同。将照片作为组加载。</li>
	<li>取消选中图像中的单个框以使强度正常化。</li>
	<li>选择光子模式来分析强度。通过在ROI 工具中选择 "感兴趣区域" 按钮来量化信号的强度。</li>
	<li>将 ROI 圆圈拖动到包含发光信号的区域, 然后单击 "测量" 按钮以获取信号强度。</li>
</ol>

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B., 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=Targeting+Stat3+and+Smad7+to+restore+TGF-beta+cytostatic+regulation+of+tumor+cells+in+vitro+and+in+vivo.">Targeting Stat3 and Smad7 to restore TGF-beta cytostatic regulation of tumor cells in vitro and in vivo.</a> Oncogene. 32, 2433-2441 (2013).</li><li>Reed, L. J., Muench, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Simple+Method+of+Estimating+Fifty+Per+Cent+Endpoints.">A Simple Method of Estimating Fifty Per Cent Endpoints.</a> American Journal of Epidemiology. 27, 493-497 (1938).</li><li>Schneider, C. A., Rasband, W. S., Eliceiri, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NIH+Image+to+ImageJ:+25+years+of+image+analysis.">NIH Image to ImageJ: 25 years of image analysis.</a> Nature methods. 9, 671-675 (2012).</li><li>Dennler, S., Prunier, C., Ferrand, N., Gauthier, J. M., Atfi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Jun+inhibits+transforming+growth+factor+beta-mediated+transcription+by+repressing+Smad3+transcriptional+activity.">c-Jun inhibits transforming growth factor beta-mediated transcription by repressing Smad3 transcriptional activity.</a> The Journal of biological chemistry. 275, 28858-28865 (2000).</li><li>Fink, S. P., Mikkola, D., Willson, J. K., Markowitz, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TGF-beta-induced+nuclear+localization+of+Smad2+and+Smad3+in+Smad4+null+cancer+cell+lines.">TGF-beta-induced nuclear localization of Smad2 and Smad3 in Smad4 null cancer cell lines.</a> Oncogene. 22, 1317-1323 (2003).</li><li>Zinn, K. R., 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=Noninvasive+bioluminescence+imaging+in+small+animals.">Noninvasive bioluminescence imaging in small animals.</a> ILAR J. 49, 103-115 (2008).</li><li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+cell+transfection:+the+present+and+the+future.">Mammalian cell transfection: the present and the future.</a> Analytical and Bioanalytical Chemistry. 397, 3173-3178 (2010).</li><li>Russell, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+adenovirus+and+its+vectors.">Update on adenovirus and its vectors.</a> Journal of General Virology. 81, 2573-2604 (2000).</li><li>Lee, C. S., 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=Adenovirus-Mediated+Gene+Delivery:+Potential+Applications+for+Gene+and+Cell-Based+Therapies+in+the+New+Era+of+Personalized+Medicine.">Adenovirus-Mediated Gene Delivery: Potential Applications for Gene and Cell-Based Therapies in the New Era of Personalized Medicine.</a> Genes &amp; Diseases. 4, 43-63 (2017).</li><li>Wang, I. I., Huang, I. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenovirus+technology+for+gene+manipulation+and+functional+studies.">Adenovirus technology for gene manipulation and functional studies.</a> Drug Discovery Today. 5, 10-16 (2000).</li><li>Kochanek, S., 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=A+new+adenoviral+vector:+Replacement+of+all+viral+coding+sequences+with+28+kb+of+DNA+independently+expressing+both+full-length+dystrophin+and+beta-galactosidase.">A new adenoviral vector: Replacement of all viral coding sequences with 28 kb of DNA independently expressing both full-length dystrophin and beta-galactosidase.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 5731-5736 (1996).</li><li>Wang, Q., Finer, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Second-generation+adenovirus+vectors.">Second-generation adenovirus vectors.</a> Nature Medicine. 2, 714-716 (1996).</li><li>Durham, H. D., 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=Toxicity+of+replication-defective+adenoviral+recombinants+in+dissociated+cultures+of+nervous+tissue.">Toxicity of replication-defective adenoviral recombinants in dissociated cultures of nervous tissue.</a> Experimental Neurology. 140, 14-20 (1996).</li><li>MacKenzie, K. L., Hackett, N. R., Crystal, R. G., Moore, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenoviral+vector-mediated+gene+transfer+to+primitive+human+hematopoietic+progenitor+cells:+assessment+of+transduction+and+toxicity+in+long-term+culture.">Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture.</a> Blood. 96, 100-108 (2000).</li><li>Zhang, W. W., Koch, P. E., Roth, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+wild-type+contamination+in+a+recombinant+adenoviral+preparation+by+PCR.">Detection of wild-type contamination in a recombinant adenoviral preparation by PCR.</a> Biotechniques. 18, 444-447 (1995).</li><li>Choi, Y., Chang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+vectors+for+vaccine+applications.">Viral vectors for vaccine applications.</a> Clinical and Experimental Vaccine Research. 2, 97-105 (2013).</li><li>de Cassan, S. C., 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+requirement+for+potent+adjuvants+to+enhance+the+immunogenicity+and+protective+efficacy+of+protein+vaccines+can+be+overcome+by+prior+immunization+with+a+recombinant+adenovirus.">The requirement for potent adjuvants to enhance the immunogenicity and protective efficacy of protein vaccines can be overcome by prior immunization with a recombinant adenovirus.</a> Journal of immunology. 187, 2602-2616 (2011).</li><li>Krasnykh, V. N., Mikheeva, G. V., Douglas, J. T., Curiel, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+recombinant+adenovirus+vectors+with+modified+fibers+for+altering+viral+tropism.">Generation of recombinant adenovirus vectors with modified fibers for altering viral tropism.</a> Journal of Virology. 70, 6839-6846 (1996).</li><li>Wickham, T. J., 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=Increased+in+vitro+and+in+vivo+gene+transfer+by+adenovirus+vectors+containing+chimeric+fiber+proteins.">Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.</a> Journal of Virology. 71, 8221-8229 (1997).</li><li>Smith, F., Jacoby, D., Breakefield, X. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+vectors+for+gene+delivery+to+the+nervous+system.">Virus vectors for gene delivery to the nervous system.</a> Restorative neurology and neuroscience. 8, 21-34 (1995).</li><li>Zhou, F., 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=Nuclear+receptor+NR4A1+promotes+breast+cancer+invasion+and+metastasis+by+activating+TGF-beta+signalling.">Nuclear receptor NR4A1 promotes breast cancer invasion and metastasis by activating TGF-beta signalling.</a> Nature communications. 5, 3388 (2014).</li></ol>

作者报告没有利益冲突。

我们开发了一种技术, 允许实时成像的 TGF-β/Smad3 信号在单个活细胞。利用这一新方法, 我们以前已经确定了一个子群体的细胞与动态 TGF-β/Smad3 转录活动, 与增强入侵和迁移8。该方法改进了传统的 TGF β信号检测, 如 Smad3 磷酸化和 TGF β靶向基因表达的西方印迹, 通过捕获肿瘤人群中 TGF-β/Smad3 信号的异质性, 突出单细胞生物学的重要性。此外, 单细胞成像的替代方法, 如核易位可以?...

转化生长因子β (TGF β) 是人类发展中的一项重要的细胞因子, 它通过由 II 型和 I 型受体1组成的 heterodimeric 复合物发出信号。对 II. 型受体的结合导致 I 型受体的招募和磷酸化, 进而 phosphorylates 下游 Smad2/3 蛋白2,3。这些活化 Smad2/3 蛋白绑定到 Smad4, 形成一个复杂的 translocates 进入细胞核和调控基因转录4。在恒定的情况下 TGF β/Smad 信号被严密地调控;然而, 在许多疾病中, 信号通路被解除管制, 并且经常抗原导致疾病的进展5,6,7。最近的研究表明, 细胞对 TGF β的反应是异质的, TGF β/Smad 活性细胞的亚群是以时间依赖性的方式8,9负责生物功能。TGF β/Smad 信号的常见细胞分析涉及使用固定端点化验, 只提供细胞活动的快照, 而且通常定量平均 TGF β/Smad 效应10。然而, 这些方法可能不能准确地反映 TGF-β/Smad 信号在疾病进展过程中生理状态下的分子行为。基于图像的活细胞分析捕捉细胞和生物过程的动态, 既具有空间又有时间的理解。
我们的目标是开发一种灵敏的高通量方法用于活细胞成像的 TGF β/Smad 信号使用腺病毒试剂。在这里, 我们感染了人类乳腺癌细胞系 MDA-MB-231 与腺病毒表达 Smad3 CAGA 的主题结合序列和荧光素酶 (卢克) 或 td-番茄 (td-汤姆) 报告基因。腺病毒报告系统为质粒的引入提供了一种快速、廉价的方法, 可导致癌细胞株感染率100%。腺病毒报告系统也已成功地应用于细胞系难以染与常规质粒11。在本协议中, 我们将描述一个可重现和无创的过程, 以实现 TGFβ/Smad 信号通路的活体细胞成像在体内和体外。

<table><tbody><tr><td>DMEM</td><td>ThermoFisher</td><td>1881024</td><td>Warm in 37 &amp;deg;C waterbath before use</td></tr><tr><td>Foetal Bovine Serum</td><td>Scientifix Life</td><td>FBS500-S</td><td>Heat inactivated before use</td></tr><tr><td>Recombinant Human TGF-&amp;beta;1</td><td>PEPROTECH</td><td>100-21</td><td>Aliquot in DDW to make final concentration at 10 mg/mL</td></tr><tr><td>Hoechst-33258</td><td>Tocris Bioscience</td><td>5117</td><td>Dilute in to PBS to make final concentration at 1 &amp;mu;L/mL</td></tr><tr><td>Luciferase Reporter Assay Kit</td><td>Promega</td><td>197897</td><td>Dilute 5x in PBS before use</td></tr><tr><td>Luminometer</td><td>Promega</td><td>9100-002</td><td></td></tr><tr><td>Phase contrast fluorescence microscopy</td><td>OLYMPUS</td><td>IX50</td><td></td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>5810 R</td><td></td></tr><tr><td>VivoGl Luciferin</td><td>Promega</td><td>P1041</td><td></td></tr><tr><td>IVIS Lumina III In Vivo Imaging System</td><td>PerkinElmer</td><td>CLS136334</td><td></td></tr><tr><td>0.5% Trypsin-EDTA (10x)</td><td>ThermoFisher</td><td>15400-054</td><td>Diltue to 0.05% (1x) in PBS</td></tr><tr><td>Cell Culture Lysis 5x Reagent</td><td>Promega</td><td>E153A</td><td>Dilute to 1x in DDW</td></tr><tr><td>10% Formalin</td><td>Sigma-Aldrich</td><td>F5554-4L</td><td></td></tr><tr><td>HEK 293A</td><td>ThermoFisher</td><td>R70507</td><td></td></tr><tr><td>MDA-MB-231</td><td>ATCC</td><td>CRM-HTB-26</td><td></td></tr><tr><td>PRKDC-SCID</td><td>Animal Resources Centre</td><td>SCIDF6</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>Isoflurane</td><td>Zoetis</td><td>26675-46-7</td><td></td></tr><tr><td>Ethanol</td><td>Chem-supply</td><td>EA043-10L-P</td><td></td></tr><tr><td>Refresh Night Time</td><td>Allergan</td><td>1750D</td><td>Lubricating Eye Ointment</td></tr><tr><td>&lt;strong&gt;Solution&lt;/strong&gt;</td><td></td><td></td><td>&lt;strong&gt;Composition&lt;/strong&gt;</td></tr><tr><td>Phosphate-Buffered Saline (PBS)</td><td></td><td></td><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;.2H&lt;sub&gt;2&lt;/sub&gt;O (4 mM); NaHPO&lt;sub&gt;4 &lt;/sub&gt;(16 mM); NaCl (0.12M)</td></tr><tr><td>FBS-DMEM&amp;nbsp;</td><td></td><td></td><td>5% heat inactivated FBS; 10 &amp;mu;g/mL penicillin; 100 &amp;mu;g/mL streptomycin</td></tr></tbody></table>

live cell imaging of the tgf-	&beta;/smad3 signaling pathway in vitro and in vivo using an adenovirus reporter system

转化生长因子β (TGF) 信号调节细胞稳态所需的许多重要功能, 在许多疾病, 包括癌症中普遍发现抗原。TGF β在晚期癌症进展中与转移有强烈牵连, 激活了迁移和侵袭性肿瘤细胞的一个子集。目前的信号通路分析方法侧重于端点模型, 通常试图测量生物事件后的信号, 不反映疾病的渐进性质。在这里, 我们展示了一个新的腺病毒记者系统特定的 TGF-β/Smad3 信号通路, 可以检测转录激活的活细胞。利用Ad CAGA12-Td-汤姆记者, 我们可以在24 小时内达到 100% MDA-MB-231 细胞感染率。使用荧光记者可以实时地对活单细胞进行成像, 直接识别转录活性细胞。用 TGF β刺激感染细胞, 只显示转录活性和参与特定生物学功能的细胞子集。这种方法允许在单个细胞水平高特异性和敏感性, 以增强对与 TGF β信号的生物学功能的认识。Smad3 转录活动也可以在体内实时报告通过应用一个Ad CAGA12-卢克记者。CAGA12-卢克可以用与传统稳定转染的荧光素酶细胞系相同的方式来测量。Smad3在体内植入细胞的转录活性可以通过常规的 IVIS 影像分析, 并在肿瘤进展过程中进行监测, 为 TGF β信号通路的动力学提供独特的洞察力。我们的协议描述了一个有利的报告传递系统, 允许快速高通量成像的活细胞信号通路的体外和体内。这种方法可以扩展到一系列基于图像的检测, 并呈现为基础生物学和治疗发展的敏感和可重复的方法。

活体单细胞成像的体外研究
为了准确评估 TGF-β/Smad 信号在单细胞中的活化作用, 使用腺病毒试剂, 首先确定每一个细胞系的最佳感染多样性 (语言) 是很重要的。当100% 的细胞对腺病毒感染有阳性反应时, 不存在病变或细胞毒性作用, 就确定了最佳的教学语言。为了确定这一点, 我们使用了一个组成性活跃的Ad-巨细胞病毒-T...

Watch this Scientific Journal Video about Live Cell Imaging of the TGF-	Î²/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System at JoVE.com

Live Cell Imaging of the TGF-	&beta;/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

在这里, 我们提出了一个使用腺病毒记者系统的 TGF-β/Smad3 信号活动的活细胞成像协议。该系统实时跟踪转录活性, 可用于体外和活体动物模型中的单细胞.

腺病毒报告系统 TGF β/Smad3 信号通路的活体细胞成像

Transforming Growth Factor &#946; (TGF-&#946;) signaling regulates many important functions required for cellular homeostasis and is commonly found ...

live-cell-imaging-tgf-smad3-signaling-pathway-vitro-vivo-using-an

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

10.4K 次观看.  University of Melbourne. 在这里, 我们提出了一个使用腺病毒记者系统的 TGF-β/Smad3 信号活动的活细胞成像协议。该系统实时跟踪转录活性, 可用于体外和活体动物模型中的单细胞.

视频: Live Cell Imaging of the TGF-	β/Smad3 Signaling Pathway In Vitro and In Vivo Using an Adenovirus Reporter System

Spheroid Assay to Measure TGF-&#946;-induced Invasion

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis at later stage1,2. Moreover, TGF-&beta; is frequently overexpressed in breast cancer and its expression correlates with poor prognosis and metastasis 3,4. The mechanisms by which TGF-&beta; induces invasion are not well understood.
TGF-&beta; elicits its cellular responses via TGF-&beta; type II (T&beta;RII) and type I (T&beta;RI) receptors. Upon TGF-&beta;-induced heteromeric complex formation, T&beta;RII phosphorylates the T&beta;RI. The activated T&beta;RI initiates its intracellular canonical signaling pathway by phosphorylating receptor Smads (R-Smads), i.e. Smad2 and Smad3. These activated R-Smads form heteromeric complexes with Smad4, which accumulate in the nucleus and regulate the transcription of target genes5. In addition to the previously described Smad pathway, receptor activation results in activation of several other non-Smad signaling pathways, for example Mitogen Activated Protein Kinase (MAPK) pathways6.
To study the role of TGF-&beta; in different stages of breast cancer, we made use of the MCF10A cell system. This system consists of spontaneously immortalized MCF10A1 (M1) breast epithelial cells7, the H-RAS transformed M1-derivative MCF10AneoT (M2), which produces premalignant lesions in mice8, and the M2-derivative MCF10CA1a (M4), which was established from M2 xenografts and forms high grade carcinomas with the ability to metastasize to the lung9. This MCF10A series offers the possibility to study the responses of cells with different grades of malignancy that are not biased by a different genetic background.
For the analysis of TGF-&beta;-induced invasion, we generated homotypic MCF10A spheroid cell cultures embedded in a 3D collagen matrix in vitro (Fig 1). Such models closely resemble human tumors in vivo by establishing a gradient of oxygen and nutrients, resulting in active and invasive cells on the outside and quiescent or even necrotic cells in the inside of the spheroid10. Spheroid based assays have also been shown to better recapitulate drug resistance than monolayer cultures11. This MCF10 3D model system allowed us to investigate the impact of TGF-&beta; signaling on the invasive properties of breast cells in different stages of malignancy.

An assay to quantitatively measure Transforming Growth Factor (TGF)-&#x03B2;-induced invasion in 3-dimensional collagen gels is described. This assay takes advantage of the MCF10A series of cell lines, which represent different stages of breast cancer development. This method can be adopted to be used with other cell lines and might be used to investigate other potential activators or inhibitors of invasion.

球体的检测，以测量TGF -β诱导的入侵

TGF-&beta; has opposing roles in breast cancer progression by acting as a tumor suppressor in the initial phase, but stimulating invasion and metastasis ...

科研

医学

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 T cells in vitro with TCR- and co-stimulation in the presence of TGF&beta; and IL-2. This bears enormous potential for future therapies, however, the molecules and signaling pathways that control differentiation are largely unknown.
Primary T cells can be manipulated through ectopic gene expression, but common methods fail to target the most important naive state of the T cell prior to primary antigen recognition. Here, we provide a protocol to express ectopic genes in naive CD4 T cells in vitro before inducing Treg differentiation. It applies transduction with the replication-deficient adenovirus and explains its generation and production. The adenovirus can take up large inserts (up to 7 kb) and can be equipped with promoters to achieve high and transient overexpression in T cells. It effectively transduces naive mouse T cells if they express a transgenic Coxsackie adenovirus receptor (CAR). Importantly, after infection the T cells remain naive (CD44low, CD62Lhigh) and resting (CD25-, CD69-) and can be activated and differentiated into Tregs similar to non-infected cells. Thus, this method enables manipulation of CD4 T cell differentiation from its very beginning. It ensures that ectopic gene expression is already in place when early signaling events of the initial TCR stimulation induces cellular changes that eventually lead into Treg differentiation.

Adenoviral gene transfer into naive CD4 T cells with transgenic expression of the Coxsackie adenovirus receptor enables the molecular analysis of regulatory T cell differentiation in vitro.

腺病毒转导朴素的CD4 T细胞调节性T细胞分化研究

Regulatory T cells (Tregs) are essential to provide immune tolerance to self as well as to certain foreign antigens. Tregs can be generated from naive CD4 ...

免疫与感染

Studying TGF-&#946; Signaling and TGF-&#946;-induced Epithelial-to-mesenchymal Transition in Breast Cancer and Normal Cells

Transforming growth factor-&#946; (TGF-&#946;) is a secreted multifunctional factor that plays a key role in intercellular communication. Perturbations of TGF-&#946; signaling can lead to breast cancer. TGF-&#946; elicits its effects on proliferation and differentiation via specific cell surface TGF-&#946; type I and type II receptors (i.e., T&#946;RI and T&#946;RII) that contain an intrinsic serine/threonine kinase domain. Upon TGF-&#946;-induced heteromeric complex formation, activated T&#946;RI elicits intracellular signaling by phosphorylating SMAD2 and SMAD3. These activated SMADs form heteromeric complexes with SMAD4 to regulate specific target genes, including plasminogen activation inhibitor 1 (PAI-1, encoded by the SERPINE1 gene). The induction of epithelial-to-mesenchymal transition (EMT) allows epithelial cancer cells at the primary site or during colonization at distant sites to gain an invasive phenotype and drive tumor progression. TGF-&#946; acts as a potent inducer of breast cancer invasion by driving EMT. Here, we describe systematic methods to investigate TGF-&#946; signaling and EMT responses using premalignant human MCF10A-RAS (M2) cells and mouse NMuMG epithelial cells as examples. We describe methods to determine TGF-&#946;-induced SMAD2 phosphorylation by Western blotting, SMAD3/SMAD4-dependent transcriptional activity using luciferase reporter activity and SERPINE1 target gene expression by quantitative real-time-polymerase chain reaction (qRT-PCR). In addition, methods are described to examine TGF-&#946;-induced EMT by measuring changes in morphology, epithelial and mesenchymal marker expression, filamentous actin staining and immunofluorescence staining of E-cadherin. Two selective small molecule TGF-&#946; receptor kinase inhibitors, GW788388 and SB431542, were used to block TGF-&#946;-induced SMAD2 phosphorylation, target genes and changes in EMT marker expression. Moreover, we describe the transdifferentiation of mesenchymal breast Py2T murine epithelial tumor cells into adipocytes. Methods to examine TGF-&#946;-induced signaling and EMT in breast cancer may contribute to new therapeutic approaches for breast cancer.

We describe a systematic workflow to investigate TGF-&#946; signaling and TGF-&#946;-induced EMT by studying the protein and gene expression involved in this signaling pathway. The methods include Western blotting, a luciferase reporter assay, qPCR, and immunofluorescence staining.

研究TGF-β信号和TGF-β引起的乳腺癌和正常细胞的上皮到中位过渡

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

癌症研究

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

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

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

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

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

生物化学

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

毛因神经发生期间诺奇信号动力学的实时生物发光成像

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...

发育生物学

Adenovirus-mediated Genetic Removal of Signaling Molecules in Cultured Primary Mouse Embryonic Fibroblasts

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction analysis. One particular method to achieve this conditional deletion is via the use of the Cre-loxP system. This method involves flanking the gene of interest with loxP sites, which are specific recognition sequences for the Cre recombinase protein. Exposure of the so-called floxed (flanked by loxP site) DNA to this enzyme results in a Cre-mediated recombination event at the loxP sites, and subsequent excision of the intervening gene3. Several different methods exist to administer Cre recombinase to the site of interest. In this video, we demonstrate the use of an adenovirus containing the Cre recombinase gene to infect primary mouse embryonic fibroblasts (MEFs) obtained from embryos containing a floxed Rac1 allele1. Our rationale for selecting Rac1 MEFs for our experiments is that clear morphological changes can be seen upon deletion of Rac1, due to alterations in the actin cytoskeleton2,5. 72 hours following viral transduction and Cre expression, cells were stained using the actin dye phalloidin and imaged using confocal laser scanning microscopy. It was observed that MEFs which had been exposed to the adeno-Cre virus appeared contracted and elongated in morphology compared to uninfected cells, consistent with previous reports2,5. The adenovirus method of Cre recombinase delivery is advantageous as the adeno-Cre virus is easily available, and gene deletion via Cre in nearly 100% of the cells can be achieved with optimized adenoviral infection.

In this video we use an adenovirus carrying the Cre recombinase gene to infect primary mouse embryonic fibroblasts carrying a floxed Rac1 allele.

腺病毒介导的基因去除信号分子在原代培养的小鼠胚胎成纤维

The ability to genetically remove specific components of various cell signalling cascades has been an integral tool in modern signal transduction ...

生物学

In Vivo Two-Color 2-Photon Imaging of Genetically-Tagged Reporter Cells in the Skin

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located in. Although traditional imaging techniques are useful in identifying protein interactions and morphology in fixed tissue, they are not able to give insight as to how quickly cells are able to bind and uptake proteins, and once activated how their morphology changes in vivo. In the present study, we ask 2 major questions: 1) what is the time-course of fibroblast activation via toll-like receptor-4 (TLR4) and lipopolysaccharide (LPS) interaction and 2) how do these cells behave once activated? Using 2-photon imaging, we have developed a novel technique to assess the ability of LPS-FITC to bind to its cognate receptor, TLR4, expressed on peripheral fibroblasts in the genetic reporter mouse line; FSP1cre; tdTomatolox-stop-lox&#160;in vivo. This unique approach allows researchers to create in-depth, time-lapse videos and/or pictures of proteins interacting with live cells that allows one to have an increased level of granularity in understanding how proteins can alter cellular behavior.

Morphological changes occur in immune responsive fibroblast cells following activation and promote alterations in cellular recruitment. Utilizing 2-photon imaging in conjunction with a genetically engineered Fibroblast-specific protein 1 (FSP1)-cre; tdTomato floxed-stop-floxed (TB/TB) mouse line and green fluorescently tagged lipopolysaccharide-FITC, we can illustrate highly specific uptake of lipopolysaccharide in dermal fibroblasts and morphological changes in vivo.

在 Vivo 双色 2 光子成像中,基因标记报告器细胞在皮肤中

Fibroblasts are mesenchymal cells that change their morphology upon activation, ultimately influencing the microenvironment of the tissue they are located ...

生物工程

Live Cell Imaging of Primary Rat Neonatal Cardiomyocytes Following Adenoviral and Lentiviral Transduction Using Confocal Spinning Disk Microscopy

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single procedure. Due to advances in microscope technology it is relatively easy to capture live cell images for the purpose of investigating cellular events in real time with minimal concern regarding phototoxicity to the cells. This protocol describes how to take live cell timelapse images of primary rat neonatal cardiomyocytes using a confocal spinning disk microscope following lentiviral and adenoviral transduction to modulate properties of the cell. The application of two different types of viruses makes it easier to achieve an appropriate transduction rate and expression levels for two different genes. Well focused live cell images can be obtained using the microscope&#8217;s autofocus system, which maintains stable focus for long time periods. Applying this method, the functions of exogenously engineered proteins expressed in cultured primary cells can be analyzed. Additionally, this system can be used to examine the functions of genes through the use of siRNAs as well as of chemical modulators.

This protocol describes a method of live cell imaging using primary rat neonatal cardiomyocytes following lentiviral and adenoviral transduction using confocal spinning disk microscopy. This enables detailed observations of cellular processes in living cardiomyocytes.

原代大鼠乳鼠心肌细胞的活细胞成像继腺病毒和慢病毒转导利用共聚焦旋转盘显微镜

Primary rat neonatal cardiomyocytes are useful in basic in vitro cardiovascular research because they can be easily isolated in large numbers in a single ...

Adenofection: A Method for Studying the Role of Molecular Chaperones in Cellular Morphodynamics by Depletion-Rescue Experiments

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell cytoskeletal structures. This involves the assembly-disassembly of higher-order macromolecular structures at a given time and location, a process that is particularly sensitive to perturbations caused by overexpression of proteins. Methods that can preserve protein homeostasis and maintain near-to-normal cellular morphology are highly desirable to determine the functional contribution of a protein of interest in a wide range of cellular processes. Transient depletion-rescue experiments based on RNA interference are powerful approaches to analyze protein functions and structural requirements. However, reintroduction of the target protein with minimum deviation from its physiological level is a real challenge. Here we describe a method termed adenofection that was developed to study the role of molecular chaperones and partners in the normal operation of dividing cells and the relationship with actin remodeling. HeLa cells were depleted of BAG3 with siRNA duplexes targeting the 3&#39;UTR region. GFP-tagged BAG3 proteins were reintroduced simultaneously into &#62;75% of the cells using recombinant adenoviruses coupled to transfection reagents. Adenofection enabled to express BAG3-GFP proteins at near physiological levels in HeLa cells depleted of BAG3, in the absence of a stress response. No effect was observed on the levels of endogenous Heat Shock Protein chaperones, the main stress-inducible regulators of protein homeostasis. Furthermore, by adding baculoviruses driving the expression of fluorescent markers at the time of cell transduction-transfection, we could dissect mitotic cell dynamics by time-lapse microscopic analyses with minimum perturbation of normal mitotic progression. Adenofection is applicable also to hard-to-infect mouse cells, and suitable for functional analyses of myoblast differentiation into myotubes. Thus adenofection provides a versatile method to perform structure-function analyses of proteins involved in sensitive biological processes that rely on higher-order cytoskeletal dynamics.

We describe a method for depletion-rescue experiments that preserves cellular integrity and protein homeostasis. Adenofection enables functional analyses of proteins within biological processes that rely on finely tuned actin-based dynamics, such as mitotic cell division and myogenesis, at the single-cell level.

Adenofection:用于研究分子伴侣在细胞地貌被耗尽，救援实验中的角色方法

Cellular processes such as mitosis and cell differentiation are governed by changes in cell shape that largely rely on proper remodeling of the cell ...

A Simple and Efficient Method for In Vivo Cardiac-specific Gene Manipulation by Intramyocardial Injection in Mice

Gene manipulation specifically in the heart significantly potentiate the investigation of cardiac disease pathomechanisms and their therapeutic potential. In vivo cardiac-specific gene delivery is commonly achieved by either systemic or local delivery. Systemic injection via tail vein is easy and efficient in manipulating cardiac gene expression by using recombinant adeno-associated virus 9 (AAV9). However, this method requires a relatively high amount of vector for efficient transduction, and may result in nontarget organ gene transduction. Here, we describe a simple, efficient, and time-saving method of intramyocardial injection for in vivo cardiac-specific gene manipulation in mice. Under anesthesia (without ventilation), the pectoral major and minor muscles were bluntly dissected, and the mouse heart was quickly exposed by manual externalization through a small incision at the fourth intercostal space. Subsequently, adenovirus encoding luciferase (Luc) and vitamin D receptor (VDR), or short hairpin RNA (shRNA) targeting VDR, was injected with a Hamilton syringe into the myocardium. Subsequent in vivo imaging demonstrated that luciferase was successfully overexpressed specifically in the heart. Moreover, Western blot analysis confirmed the successful overexpression or silencing of VDR in the mouse heart. Once mastered, this technique can be used for gene manipulation, as well as injection of cells or other materials such as nanogels in the mouse heart.