Journal: Nature Communications

Article Title: High-fat diet-induced upregulation of exosomal phosphatidylcholine contributes to insulin resistance

doi: 10.1038/s41467-020-20500-w

Figure Lengend Snippet: a Plasma and WAT cytokine array of mice that received CD63 + A33 + exosomes (L-Exo or H-Exo) for 14 days. b Fold change in H-Exo vs. L-Exo-induced plasma cytokine expression for all cytokines showing greater than two-fold change. Red bars show cytokines/factors known to be involved in insulin resistance. c TNF-α (left) and IL-6 (right) upregulation following treatment with H-Exo were confirmed by ELISA in plasma. Filled circle—PBS, filled triangle—L-Exo, and filled rectangle—H-Exo. d ITT performed on C57BL/6 mice that received exosomes via adoptive transfer for 14 days followed with or without macrophage depletion. Filled rectangle—macrophage-depleted mice treated with H-Exo; filled diamond—mice without macrophage depletion treated with PBS and circle—mice without macrophage depletion treated with H-Exo. e Glucose uptake assay performed on hepatocytes cultured with different concentrations of H-Exo (as indicated in the figure). f Glucose uptake assay performed on mouse hepatocytes supplemented with supernatant derived from macrophages cultured with nanoparticles derived from H-Exo total lipids (H-Exo Nano) and PC (34:2). g Supernatants from H-Exo-treated macrophages (monocytes+ 5 × 10 6 ) were preneutralized with anti-TNF-α and/or anti-IL-6 antibodies. Glucose uptake by hepatocytes cultured in the presence of preneutralized supernatant was estimated. Data are represented as the mean ± SD. One-way ANOVA with a Tukey post hoc test. * < 0.05; **** < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Cytokines were analyzed with a Proteome Profiler Mouse XL Cytokine Array Kit (R&D Systems, ARY028) as per the manufacturer’s instructions.

Techniques: Clinical Proteomics, Expressing, Enzyme-linked Immunosorbent Assay, Adoptive Transfer Assay, Cell Culture, Derivative Assay

Journal: Journal of Biological Chemistry

Article Title: Xanthine Oxidoreductase Promotes the Inflammatory State of Mononuclear Phagocytes through Effects on Chemokine Expression, Peroxisome Proliferator-activated Receptor-γ Sumoylation, and HIF-1α

doi: 10.1074/jbc.m110.150847

Figure Lengend Snippet: FIGURE 9. Inhibition of XOR activity increases levels of HIF-1 protein in rat inflammatory MNP and in PMA-differentiated U937 cells. A, I-MNP were purified from rat lungs 24 h following insufflation of Th-1 cytokines and were plated in 12-well plates at 1.0 106 cells/well and grown under normoxic conditions. Nonadherent cells were removed by washing after 1 h, and cells were treated with the indicated doses of MIG132. Cells were harvested after 6 h; whole cell lysates were prepared and Western immunoblots run with antibody to HIF-1. In addition, washed, adherent cells were exposed to MIG132 (50 M) for the indicated times, and Western immunoblots were run on whole cell lysates and probed with antibody to HIF-1. Blots were subsequently stripped and re-probed with antibody to GAPDH to control for protein loading. B, I-MNP were purified and plated as in A. Plates were placed in either nor- moxic (21% O2, 5% CO2) or hypoxic culture (1% O2, 5% CO2, 94% N). After 1 h, cells were treated with the XOR inhibitors allopurinol (Allo, 150 M), oxypuri- nol (Oxy, 150 M), or Y-700 (50 nM). One h later cells were treated with MIG132 (50 M) and grown for 6 h. Whole cell lysates were then prepared and West- ern immunoblots run with independent duplicate samples as indicated. Blots were first probed with antibody to HIF-1 and subsequently with antibody to GAPDH. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of duplicate samples. C, I-MNP were purified, plated, and grown in normoxia in the presence of MIG132 as in A. Cells were exposed to the XOR inhibitor Y-700 (50 nM) for 1 h and subsequently treated with IL-1 (10 ng/ml), IFN- (20 ng/ml), or LPS (1.0 g/ml). Whole cell lysates were pre- pared and Western immunoblots run after 24 h of exposure to cytokines. Blots were run on triplicate samples, and representative blots are shown. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of triplicate blots. D, U937 cells were plated in 12-well plates at 1 106 cells/well and treated with PMA (30 nM) for 48 h. Cells were then washed; the medium was replaced, and cells were grown under normoxic or hypoxic conditions for 1 h in the presence of 50 M MIG132. Subsequently, cells were treated with Y-700 (50 nM) for 1 h and cytokines added as above. Whole cell lysates were prepared after 24 h of exposure to cytokine/MIG132, and Western immunoblots were run sequentially with antibody to HIF-1 and GAPDH. Representative blots are shown of three independent blots for each experiment. Bands from Western immunoblots were quantitated by scanning dosimetry and normalized to the signal obtained from the GAPDH blots. Data show the mean S.D. of triplicate blots. E, I-MNP were purified and plated in normoxia as in B. Levels of VEGF were quantitated in the cell-free supernatant over a period of 48 h in culture. In addition, cells were treated independently with MIG132 in the absence or presence of allopurinol, oxypurinol, or Y-700 as in B, and levels of VEGF were measured in the cell-free supernatant 6 or 24 h after treatment. Data show the mean S.D. of three independent experiments. F, I-MNP were plated as above in the presence or absence of Y-700 (1.0 mM) and in the absence of MIG132. After 24 h, cell-free supernatants were collected and analyzed with the rat-specific proteome profiler. Experiments were performed in quadruplicate, and each cytokine was analyzed from two spots on each filter (boxed for VEGF), and thus all data reflect eight independent determinations for VEGF alone. Spots were quantitated as described by the supplier using an R&D transmission mode scanner and image analysis software from R&D. Data show the mean S.D. of light transmission signals (arbitrary units) from eight spots for VEGF only and both control and Y-700 groups and were normalized first to the mean positive control spots (A1, A2, A19, A20, D1, and D2) and subsequently to the signal obtained from the control samples, which was thereby set at 1.00. ***, p 0.02.

Article Snippet: VEGF was also measured using the rat specific proteome profiler array (catalog no. ARY008, R&D Systems, Inc.).

Techniques: Inhibition, Activity Assay, Purification, Western Blot, Control, Transmission Assay, Software, Positive Control

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-C. Histones (50 μg/ml, 24 hours) induced chemokine production and release as demonstrated with a Proteome Profiler™ Antibody Array in Hepa1-6 cells. D. Knockdown of NF-κB p65 and TLR4 (but not TLR2 and RAGE) in Hepa1-6 cells inhibited histone (50 μg/ml, 24 hours)-induced CCL9/10 release as demonstrated by ELISA assay (n=3, *, p<0.05 versus control shRNA group). E. Anti-CCL9/10 neutralizing antibody (1 mg/ml) partly inhibited histone (50 μg/ml, 24 hours)-induced Hepa1-6 cell migration (n=3, *, p<0.05).

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Ab Array, Knockdown, Enzyme-linked Immunosorbent Assay, Control, shRNA, Migration

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-B. Compared with the control group, TLR4 depletion (by using TLR4 −/− mice or TLR4 knockdown cells) or inhibition of histone release (by administration of 10 mg/kg heparin or 10 mg/kg H3 neutralizing antibody) limited the formation of lung metastasis (as shown in arrow) in mice based on tail vein injection of 3×10 6 Hepa1-6 cells (N=5 mice/group, *, p<0.05 versus control group). In contrast, control IgG (10 mg/kg) did not inhibit the formation of lung metastasis (B). C. Serum nucleosome levels were reduced after treatment with heparin in wildtype, but not in TLR4 −/− mice (N=5 mice/group, *, p<0.05 versus control group). D. Conceptual relationships between histone and tumor metastasis. Histone is a nuclear DAMP and can be released during cell injury or death. Once released, histone can promote cell migration and invasion through the TLR4-ERK-NF-κB pathway, which induces chemokine production and release.

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Control, Knockdown, Inhibition, Injection, Migration

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 5. Identification of cytokines secreted by infected iAECIIs and their effect on HL-60 cells. (A) Cytokine array analysis showing the levels of the indicated cy tokines in the medium conditioned by iAECIIs infected with HCoV-229E for 24 or 48 h (24 hpi and 48 hpi). Ctrl – uninfected control. Right panel: cytokine label legend. (B) Western blot analysis of the expression of IL-8 and ICAM-1 in HCoV-229E-infected iAECIIs. (C) Transwell migration assay showing the migration capacity of HL-60 neutrophils in response to a medium conditioned by iAECIIs infected with HCoV-229E for 48 h (48 hpi) in the absence or presence of an IL-8-neutralizing antibody (IL-8 nAb). Ctrl – uninfected control conditioned medium. Data shown as means with SD error bars, n = 3, *p < 0.005, (ANOVA). (D) Representative fluorescence microscopy images of adherent HL-60 neutrophils after incubation with infected iAECIIs in the absence or presence of an ICAM-1-neutralizing antibody (ICAM-1 nAb). Ctrl – uninfected control. (E) Cell count of adherent HL-60 neutrophils. Mean values are shown with SD error bars, n = 3, *p < 0.005.

Article Snippet: Cytokines in the conditioned medium were detected using the Human Cytokine Array Kit (#ARY005B; R&D Systems), and the test strips were moistened and activated according to the manufacturer’s user manual.

Techniques: Infection, Control, Western Blot, Expressing, Transwell Migration Assay, Migration, Fluorescence, Microscopy, Incubation, Cell Counting

Journal: Virus research

Article Title: Paracrinal regulation of neutrophil functions by coronaviral infection in iPSC-derived alveolar type II epithelial cells.

doi: 10.1016/j.virusres.2024.199391

Figure Lengend Snippet: Fig. 6. RNA-seq analysis identifies upstream cytokine pathways in the immune responses by neutrophils. (A and B) Cytoscape ClueGO networks of upregulated genes by infection of iAECIIs with HCoV-229E (A) or triggered by infected HCoV-229 conditioned medium in HL-60 cells (B). (C and D) Bubble plots showing the most enriched GO-BP terms among the genes upregulated by the infection of iAECIIs with HCoV-229E (C) or the genes upregulated in HL-60 cells by cultivation in the infected HCoV-229-conditioned medium (D). (E and F) Hierarchical clustering heatmaps showing the signatures of genes differentially regulated in infected iAECIIs (E) and conditioned medium-stimulated HL-60 cells (F). (G and H) X2K network analysis showing the kinases and transcription factors predicted to regulate differentially expressed genes in infected iAECIIs (G) and conditioned medium-stimulated HL-60 cells (H).

Article Snippet: Cytokines in the conditioned medium were detected using the Human Cytokine Array Kit (#ARY005B; R&D Systems), and the test strips were moistened and activated according to the manufacturer’s user manual.

Techniques: RNA Sequencing, Infection

Journal: Cell Death & Disease

Article Title: Zebularine regulates early stages of mESC differentiation: effect on cardiac commitment

doi: 10.1038/cddis.2013.88

Figure Lengend Snippet: Characterization of HS-181 cell line after treatment with zebularine. ( a ) RT-PCR of cardiac markers after treatment with zebularine. Myh7, Myh6, Actc, cTnI and Serca2 present more expression after treatment. ( b ) Immunostaining of treated cells to detect cardiac-specific proteins. Scale bars; 50 μ m. ( c ) Blots of Proteome Profiler Array and the resulting quantification histograms demonstrating inhibition of pluripotency marker expression and ( d ) increased levels of mesodermic proteins after zebularine treatment (black arrows)

Article Snippet: Protein expression profiles were assayed using the specific human pluripotent Stem Cell array kit ‘Proteome Profiler Array' (R&D Systems Europe, Abingdon, UK; ARY010) following the manufacturer's instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Immunostaining, Inhibition, Marker