Journal: PLoS ONE

Article Title: The Role of PPARγ in Advanced Glycation End Products-Induced Inflammatory Response in Human Chondrocytes

doi: 10.1371/journal.pone.0125776

Figure Lengend Snippet: In A, chondrocytes were pretreated with anti-RAGE (5 μg/ml) for 12 hours before AGEs (100 μg/ml) stimulation. In B, chondrocytes were pretreated with SB203580, SP600125, PD98059 (10 μM) for 30 minutes prior to AGEs (100 μg/ml) stimulation. The expression of PPARγ was quantified by real-time PCR and western blotting using β-actin as an internal control. In A, densitometric analysis for PPARγ levels corrected to β-actin is shown. All data are expressed as means ± SD and are representative of three independent experiments. *: p< 0 . 05 versus control, #: p< 0 . 05 versus AGEs treatment, &: p> 0 . 05 versus AGEs treatment.

Article Snippet: Rabbit monoclonal antibodies specific for IL-1β, NF-κB p65, PPARγ, TNF-α, IκBα, β-actin and RAGE were purchased from Cell signaling Technology (Danvers, MA, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Molecular immunology

Article Title: S100A11 regulates nasal epithelial cell remodeling and inflammation in CRSwNPs via the RAGE-mediated AMPK-STAT3 pathway.

doi: 10.1016/j.molimm.2021.09.014

Figure Lengend Snippet: Fig. 5. S100A11 depends on RAGE-mediated can activate AMPK and inhibit STAT3 signaling pathway. A. The phosphorylation levels of AMPK and STAT3 were detected by Western blot after S100A11 was overexpressed. B. The histogram shows the phosphorylation ratio of AMPK and STAT3 (pAMPK/ AMPK and pSTAT3/ STAT3). C. Western blot showed the expression of cleaved-PARP, CCND1, CCNE, Bcl-2, AMPK and STAT3 phosphorylated proteins after treatment of cells with different concentrations of rh-S100A11 protein (0~10000ug/mL) for 24 h. D. Cell immunofluorescence staining detected the expression of STAT3 phosphorylated protein after treatment with 1000ug/mL rh-S100A11 protein for 24 h (bar = 20um). E. The relative mRNA levels of Fas, Stk35, Tsc2 and Socs3 after cells were treated with 1000ug/mL rh-S100A11 protein or transfected for 24 h. F. RAGE protein inhibitors affect the phosphorylation levels of AMPK and STAT3 caused by rhS100A11. The bar graphs show quantification of the results, with each value represents the mean ± SD of three independent experiments. Statistical significance is shown using the Student’s t-test analysis, *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: Rabbit polyclonal anti− CCND1 antibody (Cat. No. 60,186-1-Ig, 1:2000 diluted), anti-RAGE antibody (Cat. No. 66,833-1-Ig, 1:2000 diluted) and anti− CCNE antibody (Cat. No. 11554− 1-AP, 1:2000 diluted for WB) were purchased from Proteintech.

Techniques: Phospho-proteomics, Western Blot, Expressing, Immunofluorescence, Staining, Transfection

Journal: bioRxiv

Article Title: Persistence of a novel regeneration-associated transitional cell state in pulmonary fibrosis

doi: 10.1101/855155

Figure Lengend Snippet: a, UMAP shows scRNA-seq data from alveolar epithelial cells in healthy and IPF lungs. b, RNA velocity analysis predicts lineage trajectories in alveolar epithelial cell populations. Arrows indicates strong RNA velocities. c, UMAP plots show the expression of indicated genes in healthy and IPF lung scRNA-seq data. d, UMAP plots show enrichment of candidate signaling pathways healthy and IPF lung scRNA-seq data. e, Heatmap showing expression of known target genes of indicated signaling pathways in AEC1, AEC2, and PATS-like state. Scale indicates z-score where red is high, and blue is low. f, Violin plots showing IPF-relevant gene expression in indicated cell types/cell states in control and IPF lungs. g-h, Co-staining for PATS-like markers in human IPF lungs. g, Quadruple immunostaining for SFN (green), HTII-280 (red), KRT17 (grey) and ACTA2 (blue) in IPF lung. White arrows indicate SFN + , HTII-280 + cells. Yellow arrowheads demonstrate SFN + , KRT17, HTII-280 + cells. h, Quantification of SFN + cells in total HTII-280 + cells. Data are from three independent experiments and are presented as mean ± s.e.m. Asterisks indicate p < 0.0001. i, Left panel shows triple immunostaining for SFN (green), HTII-280 (red) and AGER (grey) and right panel shows triple immunostaining for SFN (green), CLDN4 (red) and LGALS (grey). j, Quadruple immunostaining for senescence marker p21 (green), in combination with SFN (blue), ACTA2 (red) and KRT17 (grey). White arrows show SFN + , KRT17 + , p21 + triple positive cells surrounded by ACTA2 (red) positive cells (left panel). Quadruple immunostaining for γH2AX (green), SFN (blue), HTII-280 (grey) and ACTA2 (red) in IPF lungs (right panel). Inset indicates region of single channel images shown in left side. Scale bars in g-i indicate 100 µm. j, k, β-galactosidase staining in IPF lung. Black arrows indicate X-gal staining in epithelial cells. See Supplementary Fig. 9 for all corresponding immunostainings on control lungs. Scale bar indicates 100 μm.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181-200, 1:500), CLDN4 (Invitrogen, 36-4800, 1:200), GFP (Novos Biologicals, NB100-1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5-31424, 1:250), SFN (Invitrogen, PA5-95056, 1:250 or Proteintech, 66251-1-Ig, 1:500), ACTA2 (Sigma, C6198, 1:500), and, gamma-H2AX (R&D, 4418-APC, 1:500).

Techniques: Expressing, Staining, Immunostaining, Triple Immunostaining, Marker

Journal: bioRxiv

Article Title: Persistence of a novel regeneration-associated transitional cell state in pulmonary fibrosis

doi: 10.1101/855155

Figure Lengend Snippet: a, Hematoxylin and Eosin staining of IPF lung section. Scale bar indicates 200 µm. b-e, Immunostaining for PATS-like markers in healthy human lungs. b, Co-staining for SFN (green), CLDN4 (red) and LGALS3 (grey), c, Immunostaining for SFN (green), HTII-280 (red) and AGER (grey), d, Co-staining for SFN (blue), p21 (green), ACTA2 (red) and KRT17 (grey) and e, Immunostaining for γH2AX (green), SFN (blue), ACTA2 (red) and HTII-280 (grey). White line box in merged images indicate region of single channel images shown on right. In f and g DAPI (blue) stains nuclei. f, Immunostaining for SFN (green), TP63 (blue), HTII-280 (grey) and ACTA2 (red) in healthy (left panel) and IPF lungs (right panel). Scale bars indicate 100 µm.

Article Snippet: Primary antibodies were as follows: Pro-surfactant protein C (Millipore, ab3786, 1:500), AGER (R&D systems, MAB1179, 1:250), KRT8 (DSHB, TROMA-I, 1:50), KRT17 (NSJ, V2176; 1:250), KRT19 (DSHB, TROMA-III, 1:50), tdTomato (ORIGENE, AB8181-200, 1:500), CLDN4 (Invitrogen, 36-4800, 1:200), GFP (Novos Biologicals, NB100-1770, 1:500), LGALS3 (Cedarlane, CL8942AP, 1:500); SOX4 (Invitrogen, MA5-31424, 1:250), SFN (Invitrogen, PA5-95056, 1:250 or Proteintech, 66251-1-Ig, 1:500), ACTA2 (Sigma, C6198, 1:500), and, gamma-H2AX (R&D, 4418-APC, 1:500).

Techniques: Staining, Immunostaining

Journal: Biochimica et biophysica acta

Article Title: Involvement of the nuclear high mobility group B1 peptides released from injured hepatocytes in murine hepatic fibrogenesis.

doi: 10.1016/j.bbadis.2014.06.017

Figure Lengend Snippet: Fig. 8. Amelioration of CCl4-induced mouse liver fibrosis by anti-RAGE treatment. ICR mice received CCl4 administration twice weekly for 35 days and simultaneously with treatment of either anti-RAGE neutralizing antibodies or isotype-matched control IgG (C-IgG). (A) Sera were collected from either normal and those treated with and subjected to biochemical detection for liver function integrity, including AST and ALT. (B) qPCR was used to measure gene expression levels of COL1A1, α-SMA, and TGF-β1 in normal and fibrotic mouse livers. (C) ELISA de- tection for serum TGF-β1 levels. (D) Western blotting detection for Smad2 phosphorylaton, α-SMA, and COL1A1 proteins. (E) Representative Sirius red staining images of normal and fibrotic mouse liver sections. (F) Morphometry analysis for the Sirius red-visualized collagen deposition in mouse livers. Data are expressed as mean ± SEM. *P b 0.05 vs. normal group; #P b 0.05 vs. C-IgG group.

Article Snippet: For a prophylactic anti-RAGE therapy, an isotype-matched control IgG (Jackson ImmunoResearch Lab, West Grove, PA) and an anti-RAGE neutralizing antibody (R&D Systems) were intraperitoneally administrated twice weekly at dose of 10 μg per mouse (average dose 283 μg/kg body weight), starting after 1 week of CCl4 injection.

Techniques: Control, Gene Expression, Enzyme-linked Immunosorbent Assay, Western Blot, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Increased frequency and proinflammatory signature of CD48 high S100A12 + macrophages in rheumatoid arthritis synovium. A UMAP visualization of synovial macrophages clustered into nine subpopulations on the basis of scRNA-seq data. B Disease-stratified analysis showing an increased proportion of CD48 high S100A12 + macrophages in the RA synovium compared with those in the UA, OA, and HC groups. C UMAP plots depicting S100A12 expression intensity across macrophage subclusters in different disease states. D Gene Ontology (GO) enrichment of biological processes in CD48 high S100A12 + marker genes, highlighting enrichment for defense response activation, cytokine production, and leukocyte migration. E Representative images of CD68 and S100A12 immunofluorescence staining in knee synovial tissues from RA and OA patients. Scale bar: 100 μm

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Expressing, Marker, Activation Assay, Migration, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: IRF7 is a specific transcriptional regulator of CD48 high S100A12 + macrophages. A Venn diagram showing overlapping transcription factors (TFs) identified by triplicate SCENIC analyses, with the CD48 high S100A12 + subcluster enriched for NFIL3, TGIF1, FOSL2, IRF7, and STAT1. B Heatmap of regulon activity scores (RASs) for TFs across macrophage subclusters. C Ranking of TFs in CD48 high S100A12 + macrophages by the regulon specificity score (RSS, calculated via Jensen‒Shannon divergence). D UMAP dimensionality reduction of TF activity profiles across subclusters. E – F UMAP plots highlighting spatial overlap between the CD48 high S100A12 + subcluster. ( E ) and cells with elevated IRF7 regulon activity ( F ). G Representative images of immunofluorescence staining for CD68 and IRF7 in knee synovial tissues from RA and OA patients. Scale bar: 100 μm

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Activity Assay, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: IRF7 directly regulates downstream inflammatory genes in M1 macrophages. A ChIP-seq peak heatmaps showing increased IRF7 binding to promoter/enhancer regions in LPS-stimulated M1 macrophages. B Venn diagram of 108 overlapping genes from the IRF7 ChIP-seq data and the SCENIC-predicted target genes. C Reactome pathway enrichment of IRF7-regulated genes, highlighting the involvement of NF-κB, TNF, and Toll-like receptor signalling (key genes: IL-1β, FOS, NF-κB1, PTGS2, and CXCL10). D Bulk RNA-seq heatmap showing the upregulation of IRF7 and target genes in M1-polarized macrophages ( GSE130011 , GSE154346 ). E UMAP plots of NFKB1, PTGS2, IL1B, and CXCL10 expression in the CD48 high S100A12 + subcluster. F RT‒qPCR analysis of IRF7 and M1 marker genes in siRNA-treated macrophages (performed in triplicate, with 3 distinct patient sources used for each repetition). G – H Western blot validation of IRF7 and downstream protein expression following IRF7 knockdown in M1-polarized macrophages (performed in triplicate, with 3 distinct patient sources used for each repetition). Statistical significance: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way ANOVA with the Bonferroni post hoc correction)

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: ChIP-sequencing, Binding Assay, RNA Sequencing, Expressing, Marker, Western Blot, Biomarker Discovery, Knockdown

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Local IRF7 knockdown alters the immune cell composition in CIA mice. A Schematic of intra-articular IRF7 siRNA treatment in collagen-induced arthritis (CIA) model mice. B – C Flow cytometry analysis of the CD86 and CD206 mean fluorescence intensities (MFIs) in F4/80 + macrophages from ankle joints ( n = 6). CD86: NC: 1241 ± 265.4, si-IRF7: 2469 ± 390.3, positive: 3489 ± 570.9, si-mock: 3689 ± 370.1. CD206: NC: 2225 ± 225.9, si-IRF7: 3395 ± 369.4, positive: 978.2 ± 147.9, si-mock: 2022 ± 170.6. D – E Frequencies of Foxp3 + Tregs among CD3 + CD4 + T cells ( n = 6). NC: 1.66% ± 0.15%, si-IRF7: 3.27% ± 0.28%, positive: 0.47% ± 0.17%, si-mock: 1.10% ± 0.22%. F Immunofluorescence staining for S100A12 + inflammatory macrophages in the ankle synovium of different groups. Scale bar: 100 μm. The data are presented as the means ± SDs. Statistical significance: *** P < 0.001, **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction)

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Knockdown, Flow Cytometry, Fluorescence, Immunofluorescence, Staining

Journal: Arthritis Research & Therapy

Article Title: IRF7 orchestrates proinflammatory macrophage polarization and joint destruction in rheumatoid arthritis

doi: 10.1186/s13075-025-03708-3

Figure Lengend Snippet: Local IRF7 inhibition attenuates joint inflammation and bone erosion in CIA mice. A Representative ankle joint images on day 42 postimmunization. B H&E staining and histological staining. C - E IHC staining for CD68, S100A12, and IRF7 in the ankle synovium. Scale bar: 100 μm. F Quantification of paw thickness at the ankle joint ( n = 6 per group). 42 Days after the first immunization: NC: 8.33 ± 0.02, si-IRF7: 10.39 ± 0.54, positive: 12.08 ± 0.80, si-mock: 12.65 ± 0.57, Statistical significance: **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction). G H&E staining and histological scoring of synovial hyperplasia and inflammation ( n = 6). NC: 0.00 (0.00–0.00), si-IRF7: 1.50 (1.00–2.25), positive: 2.50 (1.75–3.00), and si-mock: 3.00 (2.75–3.00). Data are shown as medians with 25% − 75% percentiles. Statistical significance: * P < 0.05 (Kruskal‒Wallis test, followed by post hoc Dunn’s test with Bonferroni correction for multiple comparisons). H - J Semiquantitative analysis analysis of CD68, S100A12, and IRF7 expression via IHC staining via ImageJ ( n = 6). Statistical significance: **** P < 0.0001 (one-way ANOVA with Bonferroni post hoc correction)

Article Snippet: Double immunofluorescence staining was performed as follows: sections were incubated overnight at 4 °C with primary antibodies against CD68 (mouse monoclonal, 1:100 dilution; 66231-2-Ig, Proteintech, China) with S100A12 (rabbit polyclonal, 1:100 dilution; 16630-1-AP, Proteintech, China) or CD68 (1:100 dilution; 66231-2-Ig) with IRF7 (rabbit polyclonal, 1:150 dilution; 22392-1-AP, Proteintech, China).

Techniques: Inhibition, Staining, Immunohistochemistry, Expressing