Extrinsic Perturbations of Cell Physiology and Associated Regulatory Networks

描述（由申请人提供）： 数据生成目标的总体目标是产生高质量的数据，测量在不同微环境（ME）上生长的30个细胞系中的表型，蛋白质和磷酸蛋白质组学以及RNA表达。这种方法将显着提高LINCS数据矩阵提供表型，蛋白质组学和转录组数据的细胞系在独特的ME条件下。此外，这些数据将用于数据分析目标中的蜂窝网络签名的后续生成。子目标1.1将为在3，060个ME上生长的30个细胞系生成10个不同的表型终点。我们将利用微环境 微阵列（MEMA），以评估ME对每种细胞系的增殖、凋亡、分化状态、细胞结合和运动性的影响。统计分析将在子目标1.2.A（共250种情况）中的第2-6年每年确定50个重要ME进行确认。将在第二个研究中心使用MEMA技术对50种ME条件中的每一种条件进行相同终点的重新测试，以提供主要结果的独立验证。同时，将使用50 ME条件在第二组阵列上测试细胞，所述第二组阵列设计用于模拟人体组织的弹性模量，以确定对基质刚度的表型的影响。从验证目标中检测的50种条件中，每年选择30种主要和验证中心之间最一致的条件，通过反相蛋白阵列（RPPA）目标1.2.B和RNA表达分析（目标I.2.C）进行分析。RPPA的目标是使用500种经验证的抗体产生数据，这些抗体针对每个ME扰动下关键信号通路中的蛋白质和磷蛋白。RNA表达分析将使用由Broad Institute的LINCS小组开发的Luminex珠技术检查1000个关键基因，该小组将使用从ME条件下生长的每个细胞系制备的RNA为我们进行相同的分析。总之，RPPA和RNA表达分析将在2种不同弹性模量下生成150种不同ME条件的数据。所有数据都将经过精心策划，收集广泛的元数据，并提供给LINCS社区用于填充数据矩阵。

DESCRIPTION (provided by applicant): The overall goal of the Data Generation Aim is to produce high quality data measuring phenotypes, protein and phosphoproteomics, and RNA expression in 30 cell lines grown on diverse microenvironments (ME). This approach will significantly enhance the LINCS data matrix by providing phenotypic, proteomic, and transcriptomic data for cell lines under unique ME conditions. Furthermore, the data will be utilized for subsequent generation of cellular network signatures in the Data Analysis Aim. Sub aim 1.1 will be generate 10 different phenotypic endpoints for 30 cell lines grown on 3,060 MEs. We will utilize Microenvironment Microarrays (MEMA) to assess the effects of the ME on proliferation, apoptosis, differentiation state, cell binding, and motility in each cell line. Statistical analysis will identify 50 significnt MEs for validation each year in years 2-6 in sub aim 1.2.A (total of 250 conditions). Each of the 50 ME conditions will be re-tested with the same endpoints at a second site using MEMA technology to provide independent validation of the primary results. At the same time, the cells will be tested using the 50 ME conditions on a second set of arrays designed to model the elastic modulus of human tissues to determine the effects on phenotypes of the matrix stiffness. From the 50 conditions tested in the validation aims, 30 of the most concordant between the primary and validation sites will be selected annually for analysis by Reverse Phase Protein Array (RPPA) aim 1.2.B and RNA expression analysis (aim I.2.C.). The RPPA aim will produce data using 500 validated antibodies against proteins and phosphoproteins in key signaling pathways under each of the ME perturbations. The RNA expression analysis will examine 1000 key genes using Luminex bead technology developed by the LINCS group at the Broad Institute, who will perform the same analysis for us using RNA prepared from each of the cell lines grown under the ME conditions. In total, the RPPA and RNA expression analysis will generate data for 150 different ME conditions under 2 different elastic moduli. All data will be carefully curated, with extensive metadata collected, and provided to the LINCS community for populating the data matrix.