Mice osteoclast-derived apoptotic bodies LC-MS/MS
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus
SUBMITTER: 
Huaimin Wang
PROVIDER: PXD069800 | iProX | Wed Oct 22 00:00:00 BST 2025
REPOSITORIES: iProX
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Project description:Purpose: The goals of this study are to describe the RNA contents in apoptotic bodies (ABs) derived from osteoclasts at different stages and to compare the differences among those ABs from difference cell stages. The biological function of these ABs are also predicted and evaluated. Methods: Primary bone marrow cells were isolated and treated with M-CSF (30ng/mL) to generate bone marrow macrophages (BMMs). BMMs were then used to generate preosteoclast (pOC) stimulated by M-CSF (30ng/mL) together with RANKL (100ng/mL) for 24 h and mature osteoclast (mOC) stimulated by M-CSF (30ng/mL) together with RANKL (100ng/mL) for 72 h or more. RNA profiles of BMM, pOC and mOC were generated by deep sequencing, using Illumina NovaSeq 6000. Results: After acquiring of clean reads, we used Hisat2 for spliced mapping. We mapped about 40 million sequence reads per sample to the mouse genome (GRCm38). Saturation rate analysis was then performed evaluating the gene coverage rate along with read depth increase. The transcripts mapping to difference functional region of the genome was then analyzed. Circos software was used to map the consistency of samples with mouse genome on a chromosomal level. The quantification of transcript intensity was analyzed using Fragments Per Kilobase of exon modelper Million mapped fragments (FPKM). Conclusions: Our study represents the first high-throughput deep RAN sequencing data of cell derived apoptotic bodies. The biological prediction and analysis also revealed that ABs derived from osteoclast of different stages have distinct biological role. Followed by low throughput validation and detailed experiments, we identified that pOC derived ABs are more pro-angiogenic while mOC derived ABs are more pro-osteogenic.
2019-06-06 | GSE132230 | GEO
Project description:To survey the proteome of osteoclast secretory lysosomes, we used superparamagnetic iron oxide nanoparticles (SPIONs) to enrich for these endo-lysosomal-related organelles from murine osteoclast cultures. Briefly, large scale murine bone marrow monocyte (BMM)-derived osteoclast cultures were ‘pulsed’ with SPIONs to encourage uptake into endosomes and then ‘chased’ into secretory lysosomes upon the convergence of SPION-loaded endosomes with lysosomes and secretory pathways. Following the ‘pulse-chase’, osteoclasts were homogenized, SPION-loaded organelles captured-from post-nuclear supernatants using magnetic columns, and enriched organelles eluted and processed for 1D in-gel digestion and mass spectrometry.
2023-01-25 | PXD037006 | Pride
Project description:To survey the proteome of osteoclast secretory lysosomes, we used superparamagnetic iron oxide nanoparticles (SPIONs) to enrich for these endo-lysosomal-related organelles from murine osteoclast cultures. Briefly, large scale murine bone marrow monocyte (BMM)-derived osteoclast cultures were ‘pulsed’ with SPIONs to encourage uptake into endosomes and then ‘chased’ into secretory lysosomes upon the convergence of SPION-loaded endosomes with lysosomes and secretory pathways. Following the ‘pulse-chase’, osteoclasts were homogenized, SPION-loaded organelles captured-from post-nuclear supernatants using magnetic columns, and enriched organelles eluted and processed for 1D in-gel digestion and mass spectrometry.
2023-01-25 | PXD037813 | Pride
Project description:Rational design of nanocarriers for drug delivery approaches requires an unbiased knowledge of uptake mechanisms and intracellular trafficking pathways. Here we dissected these processes using a quantitative proteomics approach. We isolated intracellular vesicles containing superparamagnetic iron oxide polystyrene nanoparticles and analyzed their protein composition by label free quantitative mass spectrometry. The proteomic snapshot of organelle marker proteins revealed that an atypical macropinocytic-like mechanism mediated the entry of nanoparticles. We show that the entry mechanism is controlled by actin reorganization, atypical macropinocytic signaling and ADP-ribosylation factor 1. Additionally, our proteomics data demonstrated a central role for multivesicular bodies and multilamellar lysosomes in trafficking and final nanoparticle storage. This was confirmed by confocal microscopy and cryo-TEM measurements. By quantitatively analyzing the protein composition of nanoparticle-containing vesicles, our study clearly defines the routes of nanoparticle entry, intracellular trafficking and the proteomic milieu of a nanoparticle-containing vesicle.
2014-09-25 | PXD001175 | Pride
Project description:Increasing evidences are revealing the important biological functions of apoptotic bodies (ABs). Here we identified the proteomic profiles of ABs from osteoclasts of distinct differentiation stages and investigated the potential functions. ABs were isolated from apoptotic bone marrow macrophages (BMMs), pre-osteoclasts (pOCs) or mature osteoclasts (mOCs). Proteomic signature analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) followed by bioinformatic analysis showed that proteomic signatures of ABs exhibited high similarities with the parental cells. Functionally, pOC-ABs induced endothelial progenitor cell (EPC) differentiation and increased CD31hiEmcnhi endothelial cell formation in a murine bone defect model via PDGF-BB. mOC-ABs induced mesenchymal stem cell (MSC) osteogenic differentiation and promoted bone formation via RANKL reverse signaling. Together, our results mapped the detailed proteomic landscapes of osteoclast derived ABs and demonstrated that the biological functions of ABs are largely inherited from corresponding parental cells, suggesting a novel extended intercellular regulation after cell apoptosis.
2021-09-08 | PXD017245 | Pride
Project description:Bone infection represents a pathological intersection of immunity and skeletal remodeling, where inflammatory responses drive excessive bone loss. Neutrophils dominate the early immune infiltration during infection, yet how their fate within infected bone marrow influences disease progression remains poorly understood. Here, we identify neutrophil-derived apoptotic bodies (Neut-ABs) as critical mediators that preserve bone integrity during infectious osteomyelitis. Bone infection triggers extensive neutrophil apoptosis, resulting in the accumulation of Neut-ABs. Integrated transcriptomic and proteomic profiling of these Neut-ABs revealed significant enrichment of TLR2 and sialyltransferase ST3GAL1, and lectin blotting confirmed that TLR2 is modified with α2,3-linked sialic acids. Mechanistically, α2,3-sialylated TLR2 on Neut-ABs functions as a molecular decoy that competitively interferes with Siglec15–TLR2 interactions in osteoclast precursors, thereby preventing precursor fusion and suppressing bone-resorptive activity. This inhibitory effect requires both the formation and sialylation of Neut-ABs, as it was abolished in Fas-deficient (MRL/Lpr) mice, upon neuraminidase-mediated desialylation, or when using Neut-ABs derived from Tlr2⁻/⁻ mice. Therapeutic delivery of Neut-ABs markedly attenuates osteolysis in infected mice without altering bacterial clearance. These findings uncover a neutrophil-to-bone apoptotic signaling axis in which sialylated vesicular decoys selectively suppress osteoclast activation, illuminating a new paradigm linking innate immune turnover to skeletal homeostasis.
2025-10-22 | PXD069804 |
Project description:Extracellular vesicles (EVs) are mammalian cell-derived nano-scale structures enclosed by a lipid bilayer that were previously considered to be cell debris with little biological value. However, EVs are now recognized to possess biological function, acting as a packaging, transport and delivery mechanism by which functional molecules (i.e. miRNAs) can be transferred to target cells over some distance. To examine the miRNA from keratinocyte-derived EVs, we isolated three distinct populations of EVs from both HaCaT and primary human keratinocytes (PKCs) and characterized their biophysical, biochemical and functional features by using microscopy, immunoblotting, nanoparticle tracking, and next generation sequencing. We identified 1048; 906; and, 704 miRNAs, respectively, in apoptotic bodies (APs), microvesicles (MVs) and exosomes (EXs) released from HaCaT, and 608; 506; and, 622 miRNAs in APs, MVs and EXs released from PKCs. In which, there were 623 and 437 identified miRNAs common to three HaCaT-derived EVs and PKC-derived EVs, respectively. In addition, we found hundreds of exosomal miRNAs that were previously un-reported and that differences in the abundance levels of the identified EV miRNAs could discriminate between the three EV populations. These data contribute to EV-identified miRNA database, especially keratinocyte-derived EV miRNA content.
2019-05-20 | GSE106453 | GEO
Project description:To survey the proteomic differences between WT and Slc37a2 knockout osteoclasts at the whole cell and secretory lysosome levels, we used superparamagnetic iron oxide nanoparticles (SPIONs) to enrich for these endo-lysosomal-related organelles from murine osteoclast cultures. Briefly, large scale murine bone marrow monocyte (BMM)-derived osteoclast cultures were ‘pulsed’ with SPIONs to encourage uptake into endosomes and then ‘chased’ into secretory lysosomes upon the convergence of SPION-loaded endosomes with lysosomes and secretory pathways. Following the ‘pulse-chase’, osteoclasts were homogenized, SPION-loaded organelles captured-from post-nuclear supernatants using magnetic columns, and enriched organelles as well as homogenates eluted and processed for 1D in-gel digestion and mass spectrometry. The samples presented here correspond to the proteome of WT and Slc37a2 knockout homogenates with secretory lysosome proteomes have been shared in another submission.
2023-01-25 | PXD037910 | Pride
Project description:Maintaining the homeostasis of immune ecological niches such as macrophages is essential for skin regeneration. In this study, apoptotic bodies (ABs) with intact bioactive substances of ADSC were constructed by biosynthesis. Further, injectable apoptotic bodies-GelMA microspheres (ABs@GMS) were constructed by microfluidics to maintain the homeostasis of the immune ecological niche in trauma, and inhibit the secretion of argininosuccinate synthetase 1 (ASS1) by ABs-induced downregulated JAK/STAT1 signaling pathway. Consequently, the macrophages’ polarization and inflammatory response were balanced in the wound with promoted angiogenesis. This ABs-functionalized microsphere slowly released biosynthetic apoptotic vesicles that expressed C1q, C3b and other surface "eat me" signals. These vesicles can be directly recognized, phagocytosed and degraded by macrophages, and allow scavenger cells to give meaning to cell death. In vitro, ABs@GMS balanced the JAK/STAT signaling pathway in a form of positive communication from dead to living cells via apoptotic vesicles. In vivo, ABs@GMS balanced the polarization of macrophages and increased the expression of α-smooth muscle actin. In conclusion, ABs synthesized by ADSC can balance traumatic immune disorders and restore homeostasis of the wound immune microenvironment, which would be a highly promising material for macrophage-mediated wound repair.
2023-11-30 | GSE237758 | GEO
Project description:We have been interested whether peripheral tolerance can be restored by vaccination with antigen-presenting cells (APCs) loaded with apoptotic bodies. Dendritic cells (DCs) are powerful APCs that have a critical role in the initiation and progression of autoimmunity. We previously demonstrated that DCs loaded with apoptotic islet cells prevents experimental type 1 diabetes (T1D), an autoimmune disease caused by beta-cell destruction. The goal of this study is to characterize the molecular changes on gene expression -transcriptome- occurring in these DCs pulsed with apoptotic bodies. Cells from four different genetically identical NOD (Non-Obese Diabetic) mice were used to obtain DCs. DCs were loaded with apoptotic bodies from NIT-1 cell line (insulinoma from NOD mice). Unloaded DCs were used as control in four paired experiments. Moreover, transcriptome from NIT-1 apoptotic bodies was determined. Mouse Gene 1.0 ST Arrays from Affymetrix (28,853 genes) were hybridized and genes with a p-value <= 0.002, adjusted p-value <= 0.08 and fold change (FC) >= 1.38 were considered upregulated, and genes with FC <= -1.37 were considered downregulated. Results demonstrate that apoptotic cells engulfment promotes molecular changes in DCs towards tolerogenic features. Gene expression profile of DCs from NOD mice that captured NIT-1 apoptotic bodies showed a downregulation of genes involved in antigen presentation and differential expression of cytokine, chemokine, natural immunity and immunoregulation genes together with the presence of specific transcripts for islet autoantigens. Molecular changes of DCs caused by the uptake of apoptotic bodies are key factors to induce antigen-specific peripheral tolerance.
2013-05-22 | E-MEXP-3374 | biostudies-arrayexpress