Project description:Using a newly-developed workflow for quantitative newly synthesized proteome analysis (QuaNPA), featuring automated sample processing and multiplexed DIA (plexDIA) analysis, changes in the newly synthesized proteome of IFN-gamma treated Hela cells were monitored over time.
Project description:To analyse the genome-wide impact of inactivation of the ATAC or SAGA coactivator complexes on RNA polymerase II (Pol II) transcription, we purified newly synthesized RNA from mutant mouse embryonic stem (ES) cell lines in which subunits of ATAC (Yeats2, Zzz3) or SAGA (Supt7l) are inactivated or depleted. We also performed this analysis in mutant cell lines in which a subunit (Tada3) of the shared histone acetyltransferase activity of these two complexes is depleted. Newly synthesized RNA was purified following the 4sU labelling method (more details in extract protocol). For two wildtype samples, the total RNA input was also analysed.
Project description:Innate stimulation with TLR ligands leads to the activation of various genes in macrophages and various populations of these cells may exhibit different responses. Here wanted to delineate and characterize these transcriptional responses in newly established, self-renewing, in vitro grown non-transformed lines (MPI cells) and bone marrow derived macrophages We used microarrays to detail the global programme of gene expression Newly synthesized RNA from independently established MPI lines and bone marrow derived macrophages was extracted and hybridized to Affymetrix microarrays
Project description:THP-1 macrophages were infected with four strains of Mycobacterium tuberculosis to study the temporal dynamics of newly synthesized proteins in the secretome. Temporal snapshots of secretome reflect the macrophage response to pathogenicity which in combination with intracellular events, completes the disease picture. However, such studies are compromised by limitations of quantitative proteomics. Metabolic labeling by SILAC allows a 3-plex experiment while isobaric chemical labeling by iTRAQ/TMT allows up to 8 to 10-plex respectively. This makes studying temporal proteome dynamics an intangible and elusive proposition. We have developed a new variant of hyperplexing method, combining triplex SILAC with 6-plex iTRAQ to achieve 18-plex quantitation in a single MS run. THP-1 macrophages were infected with H37Ra, H37Rv, BND433 and JAL2287 and the newly synthesized secreted host proteins were studied over six temporal frame still 30 hours post infection, at a difference of 4 hours each. For quantitation, the strains were encoded with two sets of triple SILAC- H37Ra & H37Rv in one and BND433 & JAL2287 in another with a control in each. These sets were then iTRAQ labeled to encode for temporal profiles across six time points in 6-plex iTRAQ. Effectively a 36-plex design with 4 replicates of each set, these experiments were completed within few days on the mass spectrometer. Using MaxQuant and in house developed tools and pipelines, we have analysed the data to map the temporal and strain specific dynamics of newly synthesized proteins in host. Hyperplexing enables large scale spatio-temporal systems biology studies where large number of samples can be processed simultaneously and in quantitative manner.
Project description:Energy metabolism and extracellular matrix function are closely connected to orchestrate and maintain tissue organization, but the crosstalk is poorly understood. Here, we used scRNA-seq analysis to uncover the importance of respiration for extracellular matrix homeostasis in mature cartilage. A combined approach of high-resolution single cell RNA sequencing, mass spectrometry/matrisome analysis and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. Genetic inhibition of respiration in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage showing disorganized chondrocytes and increased deposition of extracellular matrix material. scRNA-seq analysis identified a cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of extracellular matrix-related genes in nonarticular chondrocyte clusters. These changes were associated with alterations in extracellular matrix composition, a shift in the collagen/non-collagen protein content and an increase of collagen crosslinking and ECM stiffness. The results demonstrate, based on findings of the scRNA-seq analysis, that respiration is a key factor contributing to ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
Project description:Sequencing newly synthesized transcriptome in addition to the regular transcriptome in single cells enable the study of gene expression temporal dynamics during rapid chromatin and gene regulation processes. However, current single-cell newly synthesized transcriptome assays require in-house technology expertise to achieve a high cellular throughput, preventing their widespread application. Here, we develop NOTE-seq to simultaneously profile regular and newly synthesized transcriptome in single cells. NOTE-seq combines 4-thiouridine labeling of newly synthesized RNA, thiol-alkylation-based chemical conversion, and the streamlined 10X Genomics platform, offering a high cellular throughput accessible to and convenient for regular biology labs without single-cell technology expertise. Using NOTE-seq, we investigate the temporal dynamics of gene expression during early-stage T-cell activation in human Jurkat T cells and mouse naïve T cells, characterize transcription factors and regulons, and discover Fli-1 as a master transcription factor for gene regulation upon T-cell activation. Interestingly, Fli-1 level in T cells is sensitive to the treatment of camptothecin, a topoisomerase inhibitor used in cancer chemotherapy, indicating its potential complication on the immune system.
Project description:Growth plate chondrocytes are regulated by numerous factors and hormones as they mature during endochondral bone formation. Chondrocytes in the growth plate’s growth zone (GC cells) produce and export matrix vesicles (MVs) under the regulation of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3]. 1α,25(OH)2D3 secreted by the cells acts on the MV membrane, releasing its contents. This study examined the regulatory role 1α,25(OH)2D3 has over the production and packaging of microRNA into MVs by GC cells and the ability to release microRNA from MVs once produced. We treated GC cells with 1α,25(OH)2D3 and then sequenced the microRNA in the cells and MVs. We also treated MVs with 1α,25(OH)2D3 and determined if the microRNA was released. To assess whether MVs can act directly with chondrocytes and if this is regulated by 1α,25(OH)2D3, we stained MVs with a membrane dye and treated GC cells with them. 1α,25(OH)2D3 regulated the production and packaging of microRNA into matrix vesicles. MVs did not release microRNA when treated with 1α,25(OH)2D3, indicating a heterogeneous MV population or a protective factor. Stained MVs were endocytosed by GC cells and this was increased with 1α,25(OH)2D3 treatment. This study adds new regulatory roles for 1α,25(OH)2D3 with respect to packaging and transport of MV microRNAs.