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)中生长的30个细胞系的表型、蛋白质和磷酸蛋白质组学以及RNA表达。这种方法将通过在独特的ME条件下提供细胞系的表型、蛋白质组和转录组数据来显著增强LINCS数据矩阵。此外，这些数据将用于在数据分析目标中后续生成蜂窝网络签名。子目标1.1将为生长在3,060个MES上30个细胞系产生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对每个细胞系的增殖、凋亡、分化状态、细胞结合和运动的影响。统计分析将在分目标1.2.A(总共250个条件)中，在第2-6年中每年确定50个重要的MES进行验证。50个ME条件中的每一个都将使用MEMA技术在第二个站点使用相同的端点进行重新测试，以提供对初步结果的独立验证。与此同时，这些细胞将在第二组阵列上使用50ME条件进行测试，该阵列旨在模拟人体组织的弹性模数，以确定基质硬度对表型的影响。在验证AIMS中测试的50个条件中，每年将从初级和验证位点之间最一致的30个条件中选择30个进行反相蛋白质阵列(RPPA)AIM 1.2.B和RNA表达分析(AIM I.2.C.)分析。RPPA的目标将使用500个经过验证的抗体在每个ME扰动下针对关键信号通路中的蛋白质和磷蛋白产生数据。RNA表达分析将使用由布罗德研究所的Lincs小组开发的Luminex Bead技术来检查1000个关键基因，他们将使用在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。总而言之，RPPA和RNA表达分析将在2个不同弹性模数下生成150个不同ME条件的数据。所有数据都将经过仔细的整理，收集大量的元数据，并提供给LINCS社区，以填充数据矩阵。

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.