Journal: Nutrition & Diabetes

Article Title: Non-pharmacological interventions of intermittent fasting and pulsed radiofrequency energy (PRFE) combination therapy promote diabetic wound healing

doi: 10.1038/s41387-024-00344-9

Figure Lengend Snippet: The sequences of primer pairs used in qPCR.

Article Snippet: The primary antibody includes antibodies targeting VEGFA (1:1000, A12303, Abclone, China), COL1A2 (1: 2000, 14695-1-AP, Proteintech, China), HIF1A (1:1000, HY-P80704, MCE, USA), CGRP (1:1000, A5542, Abclone, China), P75 (1:1000, A19127, Abclone, China), α-Tubulin (1:10000, A19127, Abclone, China).

Techniques:

Journal: Nutrition & Diabetes

Article Title: Non-pharmacological interventions of intermittent fasting and pulsed radiofrequency energy (PRFE) combination therapy promote diabetic wound healing

doi: 10.1038/s41387-024-00344-9

Figure Lengend Snippet: A qRT-PCR analysis of the expression levels of CGRP target gene in mice wounds in Control group, PRFE group, Fasting group, and PRFE+Fasting group. n = 3 per group. B Representative western blotting and C relative quantitative analysis of CGRP in whole-cell protein extracts from mice wounds in different treatment groups. The densitometric are normalized by α-tubulin. n = 3 per group. D Representative immunohistochemical staining images and E the relative IOD of CGRP expressions of mice wounds in different groups. Scale bar= 100 μm. n = 3 per group. A , C , E One-way ANOVA combined with Bonferroni post hoc test. Data are plotted as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: The primary antibody includes antibodies targeting VEGFA (1:1000, A12303, Abclone, China), COL1A2 (1: 2000, 14695-1-AP, Proteintech, China), HIF1A (1:1000, HY-P80704, MCE, USA), CGRP (1:1000, A5542, Abclone, China), P75 (1:1000, A19127, Abclone, China), α-Tubulin (1:10000, A19127, Abclone, China).

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, Immunohistochemical staining, 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

Journal: Journal of Neuroinflammation

Article Title: Calcitonin gene-related peptide regulates spinal microglial activation through the histone H3 lysine 27 trimethylation via enhancer of zeste homolog-2 in rats with neuropathic pain

doi: 10.1186/s12974-021-02168-1

Figure Lengend Snippet: CGRP evokes increases in the expressions of EZH2 and H3K27me3 in microglia by PKA/PKC. a , b The expression of Iba1 (a marker of microglia, red) and its colocalization with CRLR, RAMP1, or CRCP staining (green) in cultured BV2 cells a and rat primary microglia b . Scale bar 40 m. c , d Western blot analyses of EZH2 and H3K27me3 expressions in BV2 cells c and rat primary microglia d with treatment of CGRP at 0, 1, 2, 4, 6, and 12 h, respectively. e Western blot analyses for EZH2 and H3K27me3 protein levels in BV2 microglial cells with co-treatment of CGRP (1 M) and GSK126 (5 nM) for 4 h. f Western blotting analyses for EZH2 and H3K27me3 protein levels in microglial cells (BV2) with treatment of CGRP peptide (1 M) for 4 h and pretreatment with 1 M forskolin (PKA activator), 3 M PKI6-22 (PKA inhibitor), 325 nM PMA (PKC activator), or 5 M chelerythrine chloride (PKC inhibitor) for 30 min. The mean optic densities of the proteins were calculated by normalizing to GAPDH. All values are expressed as the means SEMs ( n = 4). * p < 0.05 vs. controls, # p < 0.05 vs. CGRP only groups. g Shown is an example of microglial cell growth curves by RTCA. RTCA was performed to evaluate the proliferation and viability of microglial cells with continuous treatment of CGRP and co-treatment of GSK126

Article Snippet: Double-labeling immunofluorescence staining for primary antibodies against Iba1 and CRLR (Abcam), RAMP1 (Sigma-Aldrich), or CRCP (Proteintech) on coverslip-cultured microglial cells was performed.

Techniques: Expressing, Marker, Staining, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: Therapeutic interleukin-2 rewires skeletal-immune circuits to reverse postmenopausal bone loss

doi: 10.64898/2026.01.07.696511

Figure Lengend Snippet: (A) Schematics of the experiment. Rag1 -/- female mice received daily subcutaneous injections of low-dose IL-2 (30,000 I.U.) or PBS for 4 weeks, 4 weeks after ovariectomy or sham operation. (B) Representative 3D μCT images of the distal femurs. (C) Quantification of μCT images by BV/TV (bone volume per tissue volume), Tb.N (trabecular number), Tb.Sp (trabecular separation), and Tb.Th (trabecular thickness), n=7-10 per group, bar indicates mean, one-way ANOVA, ∗P<0.05, ∗∗P<0.01. (D) Representative histological images of bone sections stained with H&E (top), TRAP (middle), and anti-TRAP immunohistochemistry (bottom) to evaluate the abundance of osteoclasts on bone surface. (E) UMAP visualization of bone marrow innate lymphoid cell (ILC) subsets from scRNA-seq data (Left) with key genes identifying ILC2s and ILC1s/ILC3s subclusters (Right). (F) Split UMAP visualization showing the distribution of bone marrow ILCs in each treatment group. (G) Pie charts showing the ratio of ILC2s to ILC1s/ILC3s. (H-I) Representative flow cytometry plots (H) and the quantification of BM-ILC2s (Lin - CD45 + CD127 + ST2 + Sca-1 + ) in WT and Rag1 -/- mice (I) . (J) Volcano plot of bulk RNA-seq data from purified ILC2s showing differential expression between control and IL-2 treated groups. (K) GO Pathway enrichment analysis of ILC2s comparing OVX vs. OVX + IL-2 groups from scRNA-seq data. (L-M) FACS sorted bone marrow CD3 - B220 - NK1.1 - CD11b + cells and ILC2s (Lin - CD45 + CD127 + Sca-1 + ST2 + ) from WT, Il10 -/- , Calca -/- and Il10 -/- Calca -/- (DKO) were cocultured at indicated ratios, in the presence of 20 ng/ml M-CSF and 50 ng/ml RANKL. Osteoclast formation was evaluated by TRAP staining. Representative images (L) and quantification (M) of TRAP + multinucleated (nuclei > 3) cells. Representative of at least 3 independent experiments. Bar indicates mean, one-way ANOVA, ∗∗P<0.01, ∗∗∗P<0.001, ∗∗∗∗P<0.0001. (N) Flow cytometry analysis of peripheral blood ILC2s in healthy donors (n=27) compared to patients with osteoporosis (n=17). T-test, ∗P<0.05. (O) Pearson correlation analysis between ILC2s and clinical samples with available BMD T-score, n=24, black indicates healthy donor, red indicates osteoporosis patients.

Article Snippet: The concentrations of IL-10 and CGRP in cultured ILC2 supernatants were measured using mouse IL-10 ELISA Kit (Biolegend) and CGRP ELISA kit (Boster), according to the manufacturer’s instruction.

Techniques: Staining, Immunohistochemistry, Flow Cytometry, RNA Sequencing, Purification, Quantitative Proteomics, Control

Journal: The Journal of comparative neurology

Article Title: Members of the CUGBP Elav-like family of RNA-binding proteins are expressed in distinct populations of primary sensory neurons.

doi: 10.1002/cne.25520

Figure Lengend Snippet: FIGURE 5 CELF4 protein expression in peptidergic (CGRP+), nonpeptidergic (IB4-binding), and myelinated (NF-H+) neurons. (a–c) Fourteen-micrometer dorsal root ganglia (DRG) sections stained with CELF4 antibody, co-stained with established histological markers. Peptidergic neurons were identified with CGRP antibody, nonpeptidergic with isolectin IB4, and myelinated fibers with Neurofilament Heavy Chain (NFH) antibody. Scatter plots show distribution of fluorescence intensities of somata collated from DRG from three replicate animals, two nonoverlapping cross sections per animal (approximately 100 somata per section). Dashed lines indicate background signals levels. (d) Pie charts showing the proportion of double positive somata from each of the three stains. (e) Mean CELF4 fluorescence values. Stats = ANOVA with post hoc t-tests, n = 3 animals; *p < .05 and **p < .01.

Article Snippet: 2.3.4 Polyclonal guinea pig anti-CGRP (Fitzgerald Industries International, 20R-CP007, RRID: AB_1282813) CGRP antibody was raised in guinea pig using calcitonin gene-related peptide conjugated to BSA as the immunogen.

Techniques: Expressing, Binding Assay, Staining, Fluorescence