Project description:We classified samples and deciphered a key genes signature of intratumor heterogeneity by Principal Component Analysis and Weighted Gene Co-expression Network Analysis. At the genome level, we identified common GB copy number alterations and but a strong inter-individual molecular heterogeneity.
Project description:To identify substrates of the ubiquitinating E3 enzyme Rsp5 we applied purified Rsp5 to duplicate protein arrays. The Rsp proteins were expressed as fusion proteins to GST. We used as a control Ubr1, a RING domain containing E3 ligase We analyzed Rsp5 from S.cerevisiae on duplicate arrays, with four control chips, two without Rsp5 and two with Ubr1.
Project description:We have compared the response to TGFbeta1 in normal and v-rasHa transduced primary mouse keratinocytes using NCI cDNA microarrays. This analysis reveals that Ha-ras alters global TGFbeta1 mediated gene expression in a gene specific manner. The expression pattern of TGFbeta1 immediate early response genes and down regulation of cell cycle control genes is not altered by Ha-ras but the induction of most extracellular matrix genes is blocked. Using Smad3 null keratinocytes, we find that the majority of TGFbeta1 responsive genes in primary keratinocytes are Smad3 dependent, but patterns of transcriptional responses suggest that the ability of Ha-ras to block TGFbeta1 mediated gene expression is not dependent on Smad3. However, the combination of oncogenic ras and loss of Smad3 prevents the TGFbeta1 dependent suppression of genes associated with cell cycle progression and apoptosis observed with either genotype alone. In addition several extracellular matrix genes are super-repressed by TGFbeta1 in the Ha-ras Smad3 null keratinocytes. These data provide a genomic framework for understanding how disruptions of TGFbeta signaling and oncogenic ras cooperate to promote premalignant progression of primary keratinocytes. Keywords: time series design RNA from Balb/c primary and ras keratinocytes treated for 1, 6, 24, and 48 hours with TGFbeta1 at 1ng/ml was compared to RNA from the corresponding untreated controls. For each timepoint sample from primary and ras cells, multiple arrays (including dye swaps) were analyzed. There are 6, 7, 6, and 5 arrays analyzed for RNA from primary keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated primary cells, respectively. There are 6, 8, 7, and 5 arrays analyzed for RNA from ras keratinocytes treated for 1, 6, 24, and 48 hours as compared to RNA from untreated ras cells, respectively. There are 50 arrays analyzed for the Balb/c time-course dataset. RNA from Smad3 WT and KO primary and ras keratinocytes treated 48 hours with TGFbeta1 were compared to RNA from its untreated corresponding controls. There are 6 arrays analyzed for the Smad3 WT/KO data set. There are 56 arrays in this series.
Project description:A cell line representative of human high-grade serous ovarian cancer (HGSOC) should not only resemble its tumor of origin at the molecular level, but also demonstrate functional utility in pre-clinical investigations. Here we report the integrated proteomic analysis of 26 ovarian cancer cell lines, HGSOC tumors, immortalized ovarian surface epithelial cells, and fallopian tube epithelial cells via a single-run mass spectrometric workflow. The in-depth quantitation of > 10,000 proteins results in three distinct cell line categories: epithelial (group I), clear cell (group II), and mesenchymal (group III). We identify a 67-protein cell line signature, which separates our entire proteomic dataset, as well as a confirmatory publicly available CPTAC/TCGA tumor proteome dataset, into a predominantly epithelial and mesenchymal HGSOC tumor cluster. This proteomics-based epithelial/mesenchymal stratification of cell lines and human tumors indicates a possible origin of HGSOC either from the fallopian tube or from the ovarian surface epithelium.
Project description:We report an integrated analysis incorporating DNA copy number analyses, somatic exon mutations, mRNA expression via RNA-sequencing, and shotgun mass spectrometry analysis of protein abundance in 108 surgically resected squamous cell lung cancers (SCC) with accompanying clinical outcome, evaluation of tumor pathology, and other clinically relevant data. We identified three major subtypes of SCC at the proteomic level, with two groups associated with inflammation/immune response or oxidation-reduction biology. Inflamed tumors could be further sub-classified based on neutrophil infiltration or antigen presentation proteomes and reflected patterns of infiltrating immune cells. No gene mutations, mRNA signatures, or proteomic subclasses were associated with outcomes; however, the presence of B-cell rich tertiary lymph node structures could be associated with better patient outcomes. By integrating our proteogenomic data with publicly available RNA interference screen data, we identified TP63, PSAT1, and AKR1C3 as vulnerabilities in SCC, particularly in the redox proteomic group. This cohort and its deep molecular data serves as an important resource to better understand biology and targets associated with SCC.
Project description:Mitochondrial DNA encodes thirteen subunits of the oxidative phosphorylation (OXPHOS) system, which are synthesized inside the organelle and essential for cellular energy supply. How mitochondrial gene expression is regulated and integrated into cellular physiology is little understood. Here, we performed a high-throughput screen combining fluorescent-labelling of mitochondrial translation products with siRNA-mediated knockdown, to identify cellular kinases regulating translation. As proof of principle, the screen identified known kinases that affect mitochondrial translation, and it also revealed several kinases not yet linked to this process. Among the latter, we focused on the primarily cytosolic kinase FN3K, which localizes partially to mitochondria, to support translation. Mass spectrometric (MS) bottom-up analysis of peptide samples after FN3K-flag immunoisolation in isolated mitochondria (transfected with FN3K-flag plasmid) enabled the identification of several proteins of the mitochondrial ribosome to be interacting with FN3K. Further experiments showed that FN3K likely modulates the assembly of mitochondrial ribosomes, thereby affecting translation. Overall, our work provides a reliable approach to identify new protein functions for mitochondrial gene expression, in a high throughput manner.
Project description:Colorectal cancers (CRC) with deficient mismatch repair (dMMR) exhibit variable responses to immune checkpoint inhibitors, despite their immunogenic nature. To investigate the molecular basis of this heterogeneity, we performed integrated bulk RNA sequencing and immune repertoire profiling on tumor and adjacent normal mucosa from treatment-naïve dMMR CRC patients. Using weighted gene co-expression network analysis, we identified transcriptional modules associated with T and B cell clonality, immune-metabolic interactions, and therapeutic responsiveness. Our dataset provides a valuable resource for understanding the tumor microenvironment in dMMR CRC and supports the development of biomarkers for patient stratification beyond mismatch repair status.
Project description:To investigate the role of viral and host factors in acute liver failure, we analyzed serum and multiple liver specimens obtained at the time of liver transplantation from four well-characterized patients. We carried out an integrated clinicopathological analysis, gene and microRNA expression profiling, next-generation sequencing, antibody-displaying phage libraries, and in vitro functional analysis of HBV variants. Liver samples were obtained from 4 patients with HBV-associated acute liver failure (ALF), 10 liver donors and 7 subjects who underwent hepatic resection for liver angioma. As this is the continuation of a previous study (Title: Integrated ordination of miRNA and mRNA expression profiles; Authors: Diaz et al.) miRNA (microRNA) data are already available at the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo) under the accession number GSE62037. The present submission concerns only mRNA data. This dataset is part of the TransQST collection.
Project description:Concerted radial migration of newly born cortical projection neurons, from their birthplace to their final target lamina, is a key step in the assembly of the cerebral cortex. The cellular and molecular mechanisms regulating radial neuronal migration in vivo are however still unclear, let alone the effects and interactions with the extracellular environment. Recent evidence suggests that distinct signalling cues act cell-autonomously but differentially at particular steps during the overall migration process. Functional MADM (Mosaic Analysis with Double Markers) analyses in comparison to global knockout also indicate a significant degree of non-cell-autonomous and/or community effects in the control of cortical neuron migration. In this project, we established a MADM-based experimental strategy for the quantitative analysis of cell-autonomous gene function versus non-autonomous and/or community effects, focusing on the Cdk5r1 gene, a neuron-specific activator of Cyclin Dependent Kinase 5 known to play a role, among other functions, in neuronal migration during brain development. This dataset corresponds to a TMT-10plex based proteomics analysis of the effects of full or mosaic KO of Cdk5r1 on the neuronal proteome and aims to provide insights into its distinct cell-autonomous and non-autonomous roles in neuronal migration. This dataset and complementary experimental approaches were integrated using computational modelling to define so far unknown cell-autonomous functions of candidate signalling pathways intersecting with non-cell-autonomous effects to coordinate radial neuron migration.