Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. Recently, disorders of metabolism are thought to be the center of many diseases such as OPLL. Advanced glycation end product (AGE) are accumulated in many extracellular matrixes such as ligament fibers, and it can functions as cellular signal through its receptor (RAGE), contributing to various events such as atherosclerosis or oxidative stress. However, its role in OPLL formation is not yet known. Therefore, we performed high-through-put RNA sequencing on primary posterior longitudinal ligament cells treated with different doses of AGEs (1µM, 5µM and negative control), with or without BMP2 (1µM). mRNA profiles of Primary human posterior longitudinal ligament cells stimulated with various stimuli (Control, 1µM AGE-BSA, 5µM AGE-BSA, 1µM AGE-BSA with BMP2, 5µM AGE-BSA with BMP2) were generated by deep sequencing on Ion Proton
Project description:This project is focus on the role of Src signaling involved in translation related to invadosome formation. The aim of this project is to decipher the molecular mechanism involved in specific and local translation into invadosome.
Project description:The type I JAK inhibitor ruxolitinib is approved for therapy of MPN patients but evokes resistance with longer exposure. Several novel type I JAK inhibitors were studied and we show that they uniformly induce resistance via a shared mechanism of JAK family heterodimer formation.Here we studied the expression profiles of SET2 cell lines persistent to several different type I JAK inhibitors in comparison to naive SET2 cells or in comparison to SET2 cells with acute exposure to ruxolitinib. Analysis of RNA isolated from several type I JAK inhibitor SET2 cell lines in comparison to naïve SET2 cells
Project description:To determine the effects of depleting TIP60, CDK8, or HIF1A on the transcriptional response to hypoxia, we performed RNAseq analysis of four HCT116 colorectal carcinoma cell lines (shNT, HIF1A-/-, shTIP60 and shCDK8) in normoxic and hypoxic (24hrs, 1% O2) conditions. PolyA RNA for two independent biological replicates was purified from HCT116 cells stably expressing an shRNA against a non-targeting control (shNT), TIP60 (shTIP60) or CDK8 (shCDK8), or genetically deleted HIF1A (HIF1A-/-) subjected to 24hrs 1% O2 (hypoxia) or maintained under ambient oxygen (21%; normoxia) was sequenced on the Ion Torrent platform. Reads were aligned to the human genome and gene-level counts were used for differential expression analysis.
Project description:Ossification of the posterior longitudinal ligament (OPLL) is formed by heterogeneous ossification of posterior longitudinal ligament. The patho-mechanism of OPLL is still largely unknown. MicroRNAs are small nucleatides that function as regulators of gene expression in almost any biological process. However, few microRNAs are reported to have a role in the pathological process of OPLL. Therefore, we performed high-throughput microRNA sequencing and transcriptome sequencing of primary OPLL and PLL cells in order to decipher the interacting network of microRNAs in OPLL. MRNA and microRNA profiles were done using primary culture cells of human ossification of the posterior longitudinal ligament (OPLL) tissue and normal posterior longitudinal ligament (PLL) tissue.
Project description:To analyse protein-protein interactions on a global scale, we devised a methodology termed COLA, which uses subcellular fractionation combined with quantitative proteomics to generate multivariate subcellular localisation signatures for cellular proteins. Bootstrapped clustering was then used to match proteins with significant similarity in their localisation signatures. We utilised SILAC labeled human Retinal Pigment Epithelial-1 (RPE1) cells. Heavy and light labelled cells were fractionated using 4 parallel subcellular fractionation procedures, resulting in a total of 12 fractions. The majority of fractions come from two procedures, one using serial solubilisation (resulting in fractions for cytosol, total membrane, nuclear lumen,chromatin-bound nuclear, as well as two cytoskeletal fractions) and the other using centrifugation coupled with aqueous biphasic extraction (resulting in a total nuclear fraction, intracellular membranes fraction, plasma-membrane fraction, and a cytosol + microsomes fraction). The third fractionation procedure separated cellular protrusions using porous transwell membranes, and finally the fourth procedure separated the extracellular compartment by collecting conditioned media. Fractions from each SILAC label were then mixed with equal amounts of total cell lysate from the oposite label to generate fraction/lysate SILAC SILAC mixes. Two repicrocally labelled biological replicate experiments were performed. In addition to untreated RPE1 cells, we also analysed RPE1 cells pre-teated with the Myosin-II inhibitor Blebbistatin for 4 hours prior to fractionation to reveal changes in the protein localisation profiles upon Myosin-II inhibition, as Myosin-II is a key regulator of the cytoskeleton as well as intra-cellular trafficking (Note that the blebbistatin set is not included in the associated manuscript).