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<p>In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation) after exposure to calcium oxalate monohydrate (COM) crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography–electrospray ionization–electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ) regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β) and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β), one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin). Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.</p

Relationship of the changes in the proteome following its adaptation from 2D to 3D culture with structural and physiological properties.

<p>Relationship of the changes in the proteome following its adaptation from 2D to 3D culture with structural and physiological properties.</p

Dot plot of the ratios of protein abundance (dynamic equilibrium/exponential growth states) in various sub-cellular organelles.

<p>a) Electron transport chain: complexes I, II, III and IV and the ATPase F1 and F0. b) HnRNP: core, E complex, H complex and scaffold. c) Ribosome: cytoplasmic large 60S and small 40S subunits, mitochondrial large 39S and small 28S subunits, ribophorin linker. d) Proteasome: core, lid-base, lid and ubiquitin proteins. Error bars indicate the standard deviation of the proteins in each group and the thick grey bar indicates the average. (n = 4). For a dot blot of the spliceosome, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0106973#pone.0106973.s001" target="_blank">File S1</a>, tab P4_Dot-plot_Spliceososme.</p

Immunohistochemical staining of actin and DAPI staining of DNA.

<p>C3A cells were grown using either the classical cell culture techniques (2D) or grown as spheroids (3D). Cells from 2D cultures were fixed directly while cells grown in 3D were fixed and sectioned. a, c, e and g: HepG2/C3A exponentially growing cells (2D), b, d, f and h HepG2/C3A cells at dynamic equilibrium. a and b: phalloidin staining of filamentous actin, c and d: total actin staining; e and f same images as in a and b overlayed with DAPI staining for DNA; g and h DAPI same images as in a and b but with DAPI staining alone. All photographs were made at the same magnification: the bar in h indicates 25 µM.</p

Determination of the degree of protein oxidation (carbonylation).

<p>Proteins were extracted from cells grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques. a) OxyBlot detection of carbonylated proteins; b) loading control visualised with Coomassie Brilliant Blue total protein stain; c) Levels of protein carbonylation expressed as average % optical density: *indicates statistically significant difference (t-test, p<0.05, n = 3).</p

Experimental plan.

<p>HepG2/C3A cells were grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques and analysed by mass spectrometry, immunofluorescence or using standard assays for DNA, ATP or protein.</p

Summary of changes observed when 3D spheroid culture is compared to 2D flat culture.

<p>HepG2/C3A cells were grown using classical cell culture techniques (2D) or as spheroids in a MC2 Biotek microgravity rotating bioreactor (3D). Comparative protein levels in these two conditions were determined by mass spectrometric analysis of isotope dimethyl labelled proteins. The data is described in the text and full documentation (including extra references) is provided in the supplementary materials.</p><p>Summary of changes observed when 3D spheroid culture is compared to 2D flat culture.</p

Immunohistochemical staining of tubulin and keratin.

<p>C3A cells were grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques. Cells from 2D cultures were fixed directly while cells grown in 3D were fixed and sectioned. a, c, e and g: HepG2/C3A exponentially growing cells (2D), b, d, f and h HepG2/C3A cells at dynamic equilibrium. a and b: acetylated tubulin to highlight filaments, c and d: staining of α-tubulin; e and f keratin 8. All photographs were made at the same magnification: the bar in h indicates 25 µM.</p

Chromosome-centric Human Proteome Project (C-HPP): Chromosome 12

Following an official announcement of the Chromosome-centric Human Proteome Project (C-HPP), the Chromosome 12 (Ch12) Consortium has been established by five representative teams from five Asian countries including Thailand (Siriraj Hospital, Mahidol University), Singapore (National University of Singapore), Taiwan (Academia Sinica), Hong Kong (The Chinese University of Hong Kong), and India (Institute of Bioinformatics). We have worked closely together to extensively and systematically analyze all missing and known proteins encoded by Ch12 for their tissue/cellular/subcellular localizations. The target organs/tissues/cells include kidney, brain, gastrointestinal tissues, blood/immune cells, and stem cells. In the later phase, post-translational modifications and functional significance of Ch12-encoded proteins as well as their associations with human diseases (i.e., immune diseases, metabolic disorders, and cancers) will be defined. We have collaborated with other chromosome teams, Human Kidney and Urine Proteome Project (HKUPP), AOHUPO Membrane Proteomics Initiative, and other existing HUPO initiatives in the Biology/Disease-Based Human Proteome Project (B/D-HPP) to delineate functional roles and medical implications of Ch12-encoded proteins. The data set to be obtained from this multicountry consortium will be an important piece of the jigsaw puzzle to fulfill the missions and goals of the C-HPP and the global Human Proteome Project (HPP)