PRIDE Assigned Tags:Biomedical Dataset
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Translational regulation contributes to the secretory response of chondrocytic cells following exposure to Interleukin-1B
Chondrocytes undergo changes to their protein translational capacity during osteoarthritis progression, but a study of how disease-relevant signals affect chondrocyte protein translation at the transcriptome level has not previously been performed. In this study we describe how the inflammatory cytokine IL-1B rapidly affects protein translation in the chondrosarcoma cell line SW1353. Using ribosome profiling we demonstrate that IL-1B induced altered translation of inflammatory-associated transcripts through differential translation and the use of multiple open reading frames. Proteomic analysis of the cellular layer and the conditioned media of these cells identified that proteins which were differentially translated were most readily detected in the secretome. We have produced combined ribosome profiling and proteomic datasets which provide a valuable resource in understanding the processes that are occuring during cytokine stimulation of chondrocytic cells.
Sample Processing Protocol
SW1353 were treated for 24 hours with and without IL-1B. Media was then harvested for secretome analysis and the cell layer was harvested for proteomic analysis. SW1353 were also treated for 3 hours with and without IL-1B. Cells were then harvested for ribosome profiling analysis.
Data Processing Protocol
For secretome analysis, media was collected and subjected to in-solution trypsin digestion and LC-MS/MS was performed using an Ultimate 3000 RSLC Nano system coupled to a QExtractive mass spectrophotometer. Label-free relative quantification software PEAKS was used to analyse RAW data files for identifications with Mascot. For the cell layer, protein lysates were digested with trypsin and mass spectrometry was performed using an Ultimate 3000 RSLC Nano system coupled to a QExtractive mass spectrometer. Proteins were considered significantly changed using a -10logP score >20 (equivalent to a P value of <0.01), a fold change >2 and at least 3 unique peptides. For ribosome profiling, samples were prepared accoriding to instructions from the ARTseq Ribosome Profiling Kit (Mammalian). Successfully amplified PCR libraries were quantified using a Qubit dsDNA High Sensitivity Kit and a Bioanalyser DNA High Sensitivity Kit. The size selected pooled library was assessed by bioanalyser and subsequently by qPCR using the Illumina Library Quantification Kit on a Roche Light Cycler LC480II. FASTQ files were uploaded to the RiboGalaxy server where adapter and ribosomeal RNA sequences were removed. riboSeqR was used via RiboGalaxy to align remaning reads to the human genome (GRCh38) before performing differential translation analysis.
Ben McDermott, University of Liverpool
Dr Simon Tew, Comparative Musculoskeletal Sciences Research Group Department of Musculoskeletal Biology Institute of Ageing and Chronic Disease University of Liverpool ( lab head )
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