Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: TNF-α does not impact the levels of TNFR1 and TNFR2 on Th1 and Th17 cells. Purified CD4 + T cell subsets were stimulated with 1 µg/mL of TNF-α for 24 h and stained, for flow cytometry, with anti-human TNFR1 or TNFR2 mAbs. ( A ) Representative histograms showing the expression levels of TNFR1 and TNFR2 on Th1 and Th17 cells at baseline conditions and upon TNF-α stimulus. Solid black line histograms, isotype control; black histograms, Th1 cells; grey histograms, Th17 cells; dashed line histograms, TNF-α-treated Th1 cells; long dashed line histograms, TNF-α-treated Th17 cells. The levels of TNFR1 ( B ) and TNFR2 ( C ) were measured on purified CD4 + T cell subpopulations obtained from two to four healthy controls. Bars represent the mean values ± SD. Statistical analyses were carried out with Kruskal–Wallis followed by Dunn’s multiple comparison tests. ** p < 0.01.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Staining, Flow Cytometry, Expressing, Control, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Effect of TNFR1 or TNFR2 blockade on the production of IFN-γ and IL-17 by Th1 and Th17 cells. Purified CD4 + T lymphocyte subpopulations were incubated with neutralizing antibodies to TNFR1 or TNFR2 for 1 h prior to a 4-day stimulus with TNF-α (1 µg/mL). Intracellular staining of IFN-γ and IL-17 was assessed by flow cytometry. ( A ) Representative dot plots of Th1 and Th17 cells treated with anti-TNFR1 or anti-TNFR2 in the presence or absence of TNF-α. An isotype control was used to discard non-specific effects of the neutralizing antibodies. The fold increase in the percentages of IFN-γ ( B , D ) or IL-17 ( C , E ) producers was measured on Th1 and Th17 cells obtained from two to six healthy donors. Bars represent the mean values ± SD. Data were normalized against cells that did not receive treatment with TNF-α or TNF-α plus TNFRs blocking mAbs. For statistical analysis, Kruskal–Wallis and Dunn’s multiple comparison tests were performed. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Purification, Incubation, Staining, Flow Cytometry, Control, Blocking Assay, Comparison

Journal: International Journal of Molecular Sciences

Article Title: TNF-α Affects Signature Cytokines of Th1 and Th17 T Cell Subsets through Differential Actions on TNFR1 and TNFR2

doi: 10.3390/ijms23169306

Figure Lengend Snippet: Expression of TNFR1 and TNFR2 on Th1 and Th17 cells present in the peripheral blood of rheumatoid arthritis (RA) patients treated with adalimumab. Cell staining for flow cytometry analysis was performed on PBMC samples from healthy controls ( n = 9) and RA patients ( n = 10) before (PRE) and after (POST) treatment with adalimumab. The levels of TNFR1 ( A ) and TNFR2 ( C ) are expressed in MFI values. The frequency of TNFR1 ( B ) and TNFR2 ( D )-expressing lymphocytes are also shown. Each symbol represents data for one individual. Mean values ± SD are indicated. Significance was assessed with non-parametric Kruskal–Wallis test followed by Dunn’s multiple comparison test (for MFI data) or parametric one-way ANOVA plus Tukey’s post-test (for lymphocyte frequencies data). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Cells were then stained with a mouse anti-human TNFR1-APC mAb (clone 16803) (R&D Systems, Minneapolis, MN, USA) and a rat anti-human TNFR2-PE mAb (clone hTNFR-M1) (BD Biosciences) for 30 min at 4 °C in the dark.

Techniques: Expressing, Staining, Flow Cytometry, Comparison

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Analysis of immunohistochemical staining of RAGE and HMGB1 for TETs.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Immunohistochemical staining, Staining

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Expression of HMGB1 in thymoma type A (A), B-component type AB (B), B1 (C) is shown. Scale bar: 40 μm. On this example of B1 thymoma lymphocytes are intermingled with few tumor cells. Focus on cytoplasmic staining: a larger magnification of a WHO type B1 thymoma is shown to better display “autophagic” tumor cells (tumor cells with brownish granular cytoplasmic and absent [only hematoxylin blue] nuclear staining). Scale bar: 20 μm (D). Analogously, a type B2 (E) and B3 (F) thymoma are shown. Two examples of TC (SCC (G) and (H)) are displayed – scale bar: 40 μm. HMGB1 high mobility group box1, TETs thymic epithelial tumors, TC thymic carcinoma, SCC squamous cell carcinoma

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Expressing, Staining

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Immunohistochemistry revealed strong expression of RAGE in subcapsular cTEC of fetal (A) and adult thymus (B). Scale bar: 40 μm. For comparison the staining pattern of cytokeratins 5 and 14 – markers of epithelial cell origin on fetal thymus is shown (C). The expression pattern for HMGB1 in cTEC of fetal (D, scale bar: 40 μm) and adult thymus (E, scale bar: 20 μm) is shown. (F) A larger magnification of E is shown. Scale bar 8 μm. Arrows in F indicate HMGB1 cytoplasmic staining in cTEC. RAGE receptor for advanced glycation endproducts, HMGB1 high mobility group box1, cTEC cortical thymic epithelial cells

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Immunohistochemistry, Expressing, Comparison, Staining

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Hassall's corpuscles stained with antibodies to RAGE (A), and HMGB1 (B, small arrows point to small GC) are displayed. Scale bar: 80 μm. Similarly, RAGE (C), and HMGB1 (D) staining in GC of MG patients (scale bar: 80 μm); and RAGE expression in thymic medulla (E, arrows point to thymic medulla; scale bar: 200 μm) and macrophages (F, scale bar: 40 μm) of regular adult thymus are shown. RAGE receptor for advanced glycation endproducts, HMGB1 high mobility group box1, MG Myasthenia gravis, GC germinal center.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Concentration of sRAGE, esRAGE and HMGB1 in serum of patients.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques: Concentration Assay

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Levels of sRAGE (a), esRAGE (b) and HMGB1 (c) in sera of patients with TETs including patients with paraneoplastic MG (MG n = 11) compared to healthy volunteers are shown. To rule out the influence of MG on levels of circulating sRAGE (d) and HMGB1 (e) in patients with TETs, patients with MG were excluded from this analysis. The levels of sRAGE in non-invasive (Masaoka-Koga stage I) and invasive TETs (Masaoka-Koga stages II-IV) are shown (f). RAGE receptor for advanced glycation endproducts, sRAGE soluble RAGE, esRAGE endogenous secretory RAGE, HMGB1 high mobility group box1, TETs thymic epithelial tumors, n number, MG Myasthenia gravis.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: The levels of circulating sRAGE (a), and HMGB1 (b) in serum of patients with TETs compared to patients with thymic hyperplasia and healthy volunteers are shown. RAGE receptor for advanced glycation endproducts, sRAGE soluble RAGE, HMGB1 high mobility group box1, TET thymic epithelial tumor.

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques:

Journal: PLoS ONE

Article Title: Expression of RAGE and HMGB1 in Thymic Epithelial Tumors, Thymic Hyperplasia and Regular Thymic Morphology

doi: 10.1371/journal.pone.0094118

Figure Lengend Snippet: Patients with TETs were separated into patients with thymomas and TC, and compared to healthy volunteers. Serum concentrations of sRAGE (a) and HMGB1 (b) are shown. Patients with TC were analyzed compared to volunteers for sRAGE (c), esRAGE (d), as well as HMGB1 (e). TETs thymic epithelial tumors, RAGE receptor for advanced glycation endproducts, sRAGE soluble RAGE, esRAGE endogenous secretory RAGE, HMGB1 high mobility group box1, TC thymic carcinoma

Article Snippet: Sections were stained using affinity-purified polyclonal goat anti-human RAGE IgG (R&D Systems, Minneapolis, MN, USA) or monoclonal mouse anti-human HMGB1 IgG 2b (R&D Systems) and biotinylated anti-goat IgG or anti-mouse IgG secondary antibodies (Vector Laboratories, Burlingame, CA, USA).

Techniques:

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Representative images of U2OS and MOLM-13 cells stained live with Fc-IgG1 or the indicated Siglec-Fc reagents (red). Scale bar, 10 µm. b . Representative images of U2OS and MOLM-13 cells treated with RNase A and RNase III (RNase pool) for 30 min and stained live with the indicated Siglec-Fc reagents (red). Scale bar, 10 µm. c . Quantification of the indicated Siglec dot numbers in U2OS and MOLM-13 cells treated with RNase A and RNase III for 30 min from 3 independent experiments with the number (n) of cells analyzed noted. Statistical assessment was performed with a Student's t -test and P values are shown. Data are mean ± s.e.m. d . Representative histograms (left) from flow cytometry experiments of U2OS or MOLM-13 cells treated live with or without a sialidase pool and then analyzed for surface binding of Fc-IgG (control), Siglec-7, Siglec-11, or total surface glycan (periodate) signal. Triplicate experiments are quantified with the mean fluorescence intensity (MFI) on the right for each cell line, and two-sided Student's t -tests were used to assess statistical differences, with the fold changes noted. Data are mean ± s.e.m.

Article Snippet: For Siglecs staining in live cells, 1 μg ml −1 of recombinant human IgG1 Fc (R&D Systems), Siglec-1–Fc chimera protein (R&D Systems), Siglec-2–Fc chimera protein (R&D Systems), Siglec-3–Fc chimera protein (R&D Systems), Siglec-4–Fc chimera protein (R&D Systems), Siglec-5–Fc chimera protein (R&D Systems), Siglec-6–Fc chimera protein (R&D Systems), Siglec-7–Fc chimera protein (R&D Systems), Siglec-8–Fc chimera protein (R&D Systems), Siglec-9–Fc chimera protein (R&D Systems), Siglec-10–Fc chimera protein (R&D Systems), Siglec-11–Fc chimera protein (R&D Systems), Siglec-14–Fc chimera protein (R&D Systems) and Siglec-15–Fc chimera protein (R&D Systems) were precomplexed with 0.5 μg ml −1 of donkey anti-human IgG AF647 (ImmunoResearch) secondary antibody in FACS buffer (0.5% BSA (Sigma) in 1× PBS) for 45 min on ice.

Techniques: Staining, Flow Cytometry, Binding Assay, Control, Glycoproteomics, Fluorescence

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Representative images of U2OS and MOLM-13 cells co-stained with the indicated Siglec-Fc reagents (yellow) and 9D5 (magenta). An enlargement of the hatched box is shown, and a bright field (BF) is shown to represent the outline of the cell membrane. Scale bar, 10 µm. b . Nearest neighbor distance analysis of the Siglec pairs imaged was shown below. For each pair, the distance (nanometers, nm) from that anchor (left side protein name in the figure key) to the other pair was calculated across the indicated number of cells. These values were plotted in a density histogram. c . 10 pmol of Fc-IgG (control) or Siglec-11 was immunoprecipitated to Protein A beads. 1 µg of small RNA was then incubated to beads. RNA was extracted from the beads, purified, rPAL labeled, treated with or without RNase, and finally analyzed by Northern blot. Quantification of the ratio of RNase-sensitive sialoglycoRNA that Siglec-11 binds to IgG control is calculated across the biological triplicates. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m.

Article Snippet: For Siglecs staining in live cells, 1 μg ml −1 of recombinant human IgG1 Fc (R&D Systems), Siglec-1–Fc chimera protein (R&D Systems), Siglec-2–Fc chimera protein (R&D Systems), Siglec-3–Fc chimera protein (R&D Systems), Siglec-4–Fc chimera protein (R&D Systems), Siglec-5–Fc chimera protein (R&D Systems), Siglec-6–Fc chimera protein (R&D Systems), Siglec-7–Fc chimera protein (R&D Systems), Siglec-8–Fc chimera protein (R&D Systems), Siglec-9–Fc chimera protein (R&D Systems), Siglec-10–Fc chimera protein (R&D Systems), Siglec-11–Fc chimera protein (R&D Systems), Siglec-14–Fc chimera protein (R&D Systems) and Siglec-15–Fc chimera protein (R&D Systems) were precomplexed with 0.5 μg ml −1 of donkey anti-human IgG AF647 (ImmunoResearch) secondary antibody in FACS buffer (0.5% BSA (Sigma) in 1× PBS) for 45 min on ice.

Techniques: Staining, Membrane, Control, Immunoprecipitation, Incubation, Purification, Labeling, Northern Blot

Journal: Nature

Article Title: GlycoRNA complexed with heparan sulfate regulates VEGF-A signalling

doi: 10.1038/s41586-025-10052-8

Figure Lengend Snippet: a . Western blot analysis of the indicated amount of VEGF-A 165 . 2 independent experiments were performed. b . Lysates from HUVECs after serum starvation, treatment with or without 25 ng/mL VEGF-A 165 , and UV-crosslinking, were treated with or without RNase pool and immunoprecipitated (IP) with an anti-VEGF-A antibody (Proteintech). Immunoprecipitated samples were analyzed by Western blot using an anti-VEGF-A 165 antibody (R&D system). 3 independent experiments were performed. c . Principal component analysis (PCA) of VEGF-A 165 RIP-seq results. d . Enriched transcripts in VEGF-A 165 RIP-seq. Each dot represents a unique small non-coding RNA (ncRNA) transcript, colored by biotype as indicated in the legend. The red dotted line marks the filtering citeria: log 2 FoldChange (IP/Input) > 0.5 and adjusted p-value < 0.05. Statistical assessment was performed with a two-sided Wald test and P values are adjusted for multiple comparisons using the Benjamini-Hochbery procedure. Solid circles indicate true positive hits that were also significantly enriched relative to the IgG control (see for details). e . Abundance of enriched transcripts grouped by biotype. Color scheme matches ( d ). f . Overlap between enriched transcripts and glycoRNA defined by ManNAz-seq, grouped by ncRNA family. The p-value was calculated using a two-sided hypergeometric test with all human small ncRNAs as the background. g . Western blot analysis of the beads pre-conjugated with 5 µg of VEGF-A 165 . h . Representative images of the indicated HUVECs stained with anti-VEGF-A 165 (red). Scale bar, 10 µm. Quantification of the images with number of cells noted per biological triplicate. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m. i . Western blot analysis of whole cell lysate isolated from the indicated HUVECs. Quantification of the ratio of phosphorylated VEGFR2 (pVEGFR2) to total VEGFR2 is calculated across the biological triplicates. Statistical assessment was performed with a two-sided Student's t -test and P values are shown. Data are mean ± s.e.m. j . In vitro IP of VEGF-A 165 HS WT or HS(R/K) with small RNA and rPAL. 3 independent experiments were performed. k . MST assay of VEGF-A and small RNA treated with sialidase binding. n = 3 biologically independent repeats. Data are mean ± s.e.m. l . RNA-seq analysis of the sulfotransferases expressed from HUVECs and their FPKM values from ref. . m . EMSA analysis of the indicated VEGF-A proteins with or without the addition of rHS29.

Article Snippet: For Siglecs staining in live cells, 1 μg ml −1 of recombinant human IgG1 Fc (R&D Systems), Siglec-1–Fc chimera protein (R&D Systems), Siglec-2–Fc chimera protein (R&D Systems), Siglec-3–Fc chimera protein (R&D Systems), Siglec-4–Fc chimera protein (R&D Systems), Siglec-5–Fc chimera protein (R&D Systems), Siglec-6–Fc chimera protein (R&D Systems), Siglec-7–Fc chimera protein (R&D Systems), Siglec-8–Fc chimera protein (R&D Systems), Siglec-9–Fc chimera protein (R&D Systems), Siglec-10–Fc chimera protein (R&D Systems), Siglec-11–Fc chimera protein (R&D Systems), Siglec-14–Fc chimera protein (R&D Systems) and Siglec-15–Fc chimera protein (R&D Systems) were precomplexed with 0.5 μg ml −1 of donkey anti-human IgG AF647 (ImmunoResearch) secondary antibody in FACS buffer (0.5% BSA (Sigma) in 1× PBS) for 45 min on ice.

Techniques: Western Blot, Immunoprecipitation, Control, Staining, Isolation, In Vitro, Binding Assay, RNA Sequencing