Journal: iScience

Article Title: Oncostatin M is dispensable for the regulation of hematopoietic stem/progenitor cell traffic by neutrophils

doi: 10.1016/j.isci.2025.112646

Figure Lengend Snippet: Loss of OSM perturbs HSPC levels but not neutrophil level and circadian rhythms (A and B) Circadian levels of HSPCs (Lin - cKit + Sca1 + cells, LKS) in peripheral blood (A) and bone marrow (B) of wild-type (Wt) and Osm −/− mice at different time points. Asterisks report significance vs. ZT5 of each chart. (C and D) Osm gene expression (C) and OSM concentration in the bone marrow extracellular fluid (BMEF, D) in Wt and Osm −/− mice. (E) CXCL12 concentrations in the BMEF in Wt and Osm −/− mice. (F and G) Circadian levels of aged neutrophils in peripheral blood (F) and bone marrow (G) of Wt and Osm −/− mice at different time points. (H) Expression of selected clock genes in neutrophils and of phagocytosis genes in the BM of Wt and Osm −/− mice at Zt5 and Zt13, normalized to Ubc. Mean ± standard error is displayed in the plots. ∗ p < 0.05; ∗∗ p < 0.01; by Student’s t test (A, B, F, G, H), two-way ANOVA (C, D, E). n = 4–7 biological replicates for each strain.

Article Snippet: Commercial ELISA kits assays were used to quantify OSM (Cat. No DY495-05, Bio-Techne, Minneapolis, USA) and CXCL12 (Cat. No. MCX120, R&D Systems, Inc, Bio-Techne, Minneapolis, USA) in plasma, BMEF or culture supernatant.

Techniques: Gene Expression, Concentration Assay, Expressing

Journal: iScience

Article Title: Oncostatin M is dispensable for the regulation of hematopoietic stem/progenitor cell traffic by neutrophils

doi: 10.1016/j.isci.2025.112646

Figure Lengend Snippet: Neutrophil depletion mobilizes HSPCs (A) Depletion of peripheral blood neutrophils with anti-Ly6G antibodies in wild-type (Wt) and Osm −/− mice. (B) Depletion of bone marrow neutrophils with anti-Ly6G antibodies in Wt and Osm −/− mice. (C) HSPC (Lin - cKit + Sca1 + cells) levels in Wt and Osm −/− mice treated with anti-Ly6G antibodies or PBS control. (D) Gene expression of Cxcl12 in the bone marrow of Wt and Osm −/− mice treated with anti-Ly6G antibodies or PBS. (E) Gene expression of Cxcl12 in the bone marrow of Wt and Osm −/− mice treated with clodronate liposomes or PBS. Mean ± standard error is displayed in the plots. ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001 by two-way ANOVA (A-B-C-D-E).

Article Snippet: Commercial ELISA kits assays were used to quantify OSM (Cat. No DY495-05, Bio-Techne, Minneapolis, USA) and CXCL12 (Cat. No. MCX120, R&D Systems, Inc, Bio-Techne, Minneapolis, USA) in plasma, BMEF or culture supernatant.

Techniques: Control, Gene Expression, Liposomes

Journal: bioRxiv

Article Title: Rotary jet-spun porous microfibers as scaffolds for stem cells delivery to central nervous system injury

doi: 10.1101/239194

Figure Lengend Snippet: MSC compatibility with PRM scaffolds. (A) Characterization of PRM scaffolds by scanning electron microscopy. (B) BrdU and phalloidin immunostaining of MSCs on coverslips or PRM scaffolds. Cells were able to adhere and incorporate BrdU into DNA (proliferating cells) in either condition (n = 2, experiment in triplicates). Scale bar = 100 μm. (C) After seven days in culture, MTT assay showed similar survival of MSCs when cells were cultured on coverslips or PRM scaffolds (p > 0.05, Student’s t test, n = 5). (D) TUNEL assay showed that MSCs cultured on coverslips or PRM scaffolds did not undergo apoptosis (n = 2, experiment in triplicates). Scale bar = 100 μm. (E) MSCs cultured on coverslips or PRM scaffolds were induced to differentiate in osteocytes and adipocytes. After 3 weeks, cells were stained with Oil Red (staining of lipid vesicles in red) or Alizarin Red (staining of calcium deposits in red). MSCs cultured on PRM scaffolds preserved multipotency (n = 2, experiment in triplicates). Upper scale bar = 50 μm; lower scale bar = 200 μm. (F) PRM induced a 50% increase in CXCL12 secretion by MSCs ( p < 0.05, Student’s t test, n = 5). MSCs: mesenchymal stem cells, PRM: polymeric rough microfibers, BrdU: 5-bromo-2’-deoxyuridine, MTT: 3-(4,5-dimethylthiazol-yl)2,5-diphenyltetrazolium bromide, TUNEL: terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, DAPI: 4’,6-diamidino-2-phenylindole.

Article Snippet: CXCL 12 in conditioned media was quantified using Mouse CXCL12 DuoSet ELISA (R&D systems, Minneapolis, USA).

Techniques: Electron Microscopy, Immunostaining, MTT Assay, Cell Culture, TUNEL Assay, Staining, End Labeling

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a The mean fluorescence intensity (MFI) of CXCR4 in peripheral neutrophils from healthy controls (n = 24) and psoriasis patients (n = 54). b Correlation of the CXCR4 MFI on peripheral neutrophils with PASI in psoriasis patients (n = 25). c The proportion of circulating CD15 + CXCR4 hi neutrophils in healthy controls (n = 20) and psoriasis patients (n = 25). d Correlation of the proportions of CXCR4 hi neutrophils with PASI in psoriasis patients. The adjusted R 2 and P -values were plotted in the graph. e Representative immunoblots of total CXCR4 in circulating neutrophils from healthy controls and psoriasis patients. The relative multiple expression was counted. Blots for each antigen were processed in the same experiment in parallel. The result was repeated twice independently with similar results. f Immunofluorescence staining of CD15 (green) and CXCR4 (red) in normal and inflamed psoriatic skin. Scale bar = 50 µm. n = 10 biologically independent samples. g The proportion of CXCR4 lo vs . CXCR4 hi neutrophils in inflamed psoriatic skin (n = 30 fields from 10 patient samples). MFI of CXCR4 on peripheral neutrophils ( h ) and serum protein levels of CXCL12, IL-17A, and MPO ( i ) before and after treatment with anti-IL-17 inhibitor for 12 weeks. n = 5 biologically independent samples. Data are mean ± SD. Analyses: unpaired Student’s t-test in ( a) and ( c) ; The Spearman method in ( b ) and ( d ); Paired Student’s t-test in ( h ) and ( i ). The paired and unpaired Student’s t-test were conducted as two-sided tests. FMO, Fluorescence Minus One; HC, healthy control; MFI, mean fluorescence intensity; Pre, pre-treatment; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Fluorescence, Western Blot, Expressing, Immunofluorescence, Staining, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Representative images and quantification of different nucleus morphology of CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Scale bar = 5 µm. b Flow cytometry analysis of key immune markers between peripheral CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients (scale: log-transformed MFI). c Assessment of CXCR4 hi neutrophil survival by Annexin V and 7-AAD staining after 24 h of culture. d Measurement of ROS production by DHE fluorescence. e Quantification of NETs in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Phagocytosis of pHrodo Green E. coli by neutrophils was evaluated by immunofluorescence staining ( f ) and flow cytometry ( g ). Scale bar = 5 µm. h Degranulation of CXCR4 lo or CXCR4 hi neutrophils as assessed by CD63 expression. ELISA quantification ( i ) and representative immunoblots ( j ) for mature MMP-9 in the supernatant of CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients that cultured for 24 h. GAPDH was analyzed from corresponding neutrophils. Blots for each antigen were processed in the same experiment in parallel. k Relative mRNA expression of pro-inflammatory mediators in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Data are mean ± SD (n = 6 biologically independent samples). The immunofluorescence staining was repeated three times independently with similar results. Two-way ANOVA with Tukey’s post hoc test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; MFI, mean fluorescence intensity; MMP-9, matrix metalloprotein 9; NETs; neutrophil extracellular traps; neu, neutrophils; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Flow Cytometry, Transformation Assay, Staining, Fluorescence, Immunofluorescence, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Cell Culture, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Volcano plot showing the number of differentially expressed genes (DEGs) between CXCR4 lo and CXCR4 hi neutrophils from healthy controls (n = 7) and psoriasis patients (n = 6). b Heatmap of DEGs between psoriatic CXCR4 lo and CXCR4 hi neutrophils related to neutrophil and immune function. c Enriched GO terms between CXCR4 lo and CXCR4 hi psoriatic neutrophils. d Gene set enrichment analysis showing enrichment of genes involved in glycolysis in psoriatic CXCR4 hi neutrophils. e Flow cytometry of glycolytic markers in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. f Uptake of glucose (2-NBDG) in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. g Extracellular lactate production in CXCR4 lo and CXCR4 hi neutrophils from healthy controls and psoriasis patients. Mean ± SD (n = 6 biologically independent samples/group). Two-way ANOVA with Tukey’s post hoc test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; MFI, mean fluorescence intensity; Pso, psoriasis patients; RNA-seq, RNA sequencing. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Flow Cytometry, Control, Fluorescence, RNA Sequencing

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Representative images of adherent CXCR4 lo and CXCR4 hi neutrophils co-cultured with HMEC-1 cells. White arrows indicate HMEC-1 cells; red arrows indicate adherent neutrophils. Scale bar = 10 µm. b Relative mRNA expression of adhesion molecules in HMEC-1 cells with indicated treatment. Representative immunoblots ( c ) and qRT-PCR analysis ( d ) of tight junctions in HMEC-1 cells co-cultured with indicated treatment. e Stimulation of HMEC-1 cells with CXCR4 lo and CXCR4 hi neutrophils for 6 h, followed by analysis of leaked fluorescence intensity of FITC-dextran in a Transwell system. Representative immunoblots of HMEC-1 cells pre-incubated with LDHA inhibitor for 30 min following co-culture with psoriatic CXCR4 hi neutrophils ( f ) and qRT-PCR assessment of tight junction genes ( g ). h Analysis of released FITC-dextran in a Transwell system with HMEC-1 cells, pre-incubated with LDHA inhibitor for 30 min, followed by co-culture with psoriatic CXCR4 hi neutrophils. i Confocal images of CD31 (green) and GPR81 (purple) in psoriatic lesions (n = 6) with CD15 + CXCR4 hi neutrophils adjacent to GPR81 + vascular ECs. Scale bar = 50 µm, 20 µm. The result was repeated three times independently with similar results. HMEC-1 cells were transfected with GPR81 siRNA and subjected to indicated treatment, followed by Western blot ( j ), qRT-PCR ( k ), and FITC-dextran leakage ( l ). The immunoblotting samples shown are from the same experiment ( c , f and j ) and blots were processed in parallel. Mean ± SD (n = 6 biologically independent samples/group). Analyses: two-way ANOVA with Tukey’s post hoc test in ( b ), ( d ), ( e ), ( h ) and ( l ); One-way ANOVA with Tukey’s post hoc test in ( g ) and ( k ). One or two-way ANOVA tests were performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; HMEC-1 cells, human microvascular endothelial cells; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Cell Culture, Expressing, Western Blot, Quantitative RT-PCR, Fluorescence, Incubation, Co-Culture Assay, Transfection, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Expression of CXCR4 on neutrophils stimulated with IL-25, CXCL12, and TNF for 2 h was measured by flow cytometry. b Serum level of CXCL12 in healthy controls (n = 24) and psoriasis patients (n = 30) was detected by ELISA. c Expression of CXCR4 on neutrophils with indicated treatment was measured by flow cytometry. d Representative immunofluorescence staining of CD31 (yellow), Vimentin (green), and CXCL12 (purple) in inflamed psoriatic skin (n = 6). Scale bar = 50 µm, 20 µm. e Representative immunofluorescence staining of CD31 (yellow), CXCR4 (green), and CXCL12 (purple) in inflamed psoriatic skin (n = 6). Scale bar = 50 µm, 20 µm. QRT-PCR analysis ( f ) and flow cytometry analysis ( g ) of CXCR4 in healthy neutrophils with indicated treatments. h Representative immunofluorescence co-staining of CXCR4 (red) and LAMP1 (green) in neutrophils. Scale bar = 2 µm. i Examples of low colocalization values in a sample of CXCR4 hi and CXCR4 lo neutrophils of psoriasis patients stained for LCN2 (red) and CD63 (yellow) was visualized by ImageStream analysis. Images are from one representative experiment out of six. The scale bar indicates 7 μm. Isolated neutrophils were used for ( a ), ( c ), ( f ), and ( g ). Mean ± SD (n = 6 biologically independent samples/group). The immunofluorescence staining was repeated three times independently with similar results. Analyses: one-way ANOVA with Tukey’s post hoc test in ( a) and ( c) ; two-way ANOVA with Tukey’s post hoc test in ( f ) and ( g ); Unpaired Student’s t-test in ( b ). The unpaired Student’s t-test was conducted as two-sided tests. One or two-way ANOVA tests were performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. FMO, Fluorescence Minus One; HC, healthy control; LCN2, lipocalin 2; MFI, mean fluorescence intensity; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Expressing, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Quantitative RT-PCR, Isolation, Fluorescence, Control

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Conceptual network diagram of genes showing the relationship between CREB1 and enrichment pathways, and heatmap of top transcription factors in CXCR4 hi neutrophils. The mRNA expression of CREB1 ( b ) and protein levels of p-CREB1 (s133) ( c ) in CXCR4 lo and CXCR4 hi neutrophils. d Flow cytometry analysis of p-CREB1 (s133) in neutrophils treated with indicated stimulation. e Co-localization of CXCR4 (green) and p-CREB1 (red) in neutrophils from healthy controls and psoriasis patients. Scale bar = 2 µm. Representative staining ( f ) and Western blot ( g ) of CBP (green) and p-CREB1 (red) in neutrophils treated with indicated stimulation for 2 h. h , i Healthy neutrophils were pre-treated with CREB1 inhibitor (KG-501, 300 μM) for 1 h, followed by indicated stimulation for 2 h. Flow cytometry analysis of the expression of membrane molecules ( h ), as well as glycolysis ( i ). j Representative co-staining of DNA (Hoechst), Cit-H3 (citrullinated histone-3, red), and MPO (myeloperoxidase, green) to assess NETs formation in psoriatic neutrophils in vitro after pretreatment with KG-501 (300 μM) for 1 h. Scale bar = 20 µm. k Western blot of dHL-60 cells transfected with CREB1 siRNA and subjected to the indicated treatment. l Depiction of the CREB1 binding site in the CXCR4 promoter for ChIP assay. m The luciferase activities of wild type CXCR4 and CXCR4 with the CREB1-binding site mutant were determined by luciferase reporter gene assays in dHL-60 cells. Isolated neutrophils were used for ( b ), ( d ), ( h ), and ( i ), and whole blood was used for ( c ). Mean ± SD (n = 6 biologically independent samples). The immunofluorescence staining was repeated three times independently with similar results. Blots for each antigen were processed in the same experiment in parallel. Two-way ANOVA with Tukey’s post hoc test ( b, c, h, i , and m ) and one-way ANOVA with Tukey’s post hoc test ( d ) were performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant. HC, healthy control; MFI, mean fluorescence intensity; NETs, neutrophil extracellular traps; Pso, psoriasis patients. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Expressing, Flow Cytometry, Staining, Western Blot, Membrane, In Vitro, Transfection, Binding Assay, Luciferase, Mutagenesis, Isolation, Immunofluorescence, Control, Fluorescence

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Relative mRNA expressions of CXCR4 and CXL12 in mice tissues was evaluated by qRT-PCR. n = 6 mice. b Proportion of Ly6G + CXCR4 hi neutrophils in mice skin was determined by flow cytometry. n = 6 mice. c Serum level of CXCL12 in control and IMQ-treated mice was detected by ELISA. n = 6 mice. d Schematic diagram of mouse experimental protocol. IMQ mice were injected intraperitoneally with an anti-Ly6G antibody every other day and then injected subcutaneously with fresh isolated homologous Ly6G + CXCR4 lo or Ly6G + CXCR4 hi neutrophils daily. e Phenotype and representative H&E staining of IMQ-treated mice in indicated groups on day 5. Images are representative of six individual mouse per group. Control group was topically applied with Vaseline cream. Bar = 200 µm. n = 6 mice. f Epidermal thickness was assessed by H&E. n = 20 vision fields from 6 mice. g Representative immunofluorescence staining of CD31 (red) in inflamed skin. Scale bar = 50 µm. n = 6 mice. h Quantification of the dermal vascular area in the H&E-stained sections. n = 10 vision fields from 6 mice. i Relative mRNA expressions of inflammatory cytokines in mice tissues. n = 6 mice. The immunofluorescence staining was repeated three times independently with similar results. Mean ± SD. Analyses: unpaired Student’s t-test in ( a ), ( b ), and ( c ); One-way ANOVA with Tukey’s post hoc test in ( f ), ( h ), and ( i ). The unpaired Student’s t-test was conducted as two-sided tests. One-way ANOVA test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant; IMQ, imiquimod. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Quantitative RT-PCR, Flow Cytometry, Control, Enzyme-linked Immunosorbent Assay, Injection, Isolation, Staining, Cream, Immunofluorescence

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: a Phenotype and H&E staining of IMQ-treated mice in different treatment groups. Images are representative of six individual mice per group. The mice in the control group was topically applied with vaseline cream. Bar = 200 µm. n = 6 mice. b Epidermal thickness as assessed by H&E staining. n = 20 vision fields from 6 mice. c Proportion of Ly6G + CXCR4 hi neutrophils in inflamed skin was determined by flow cytometry. n = 6 mice. d Representative immunofluorescence staining of CD31 (red) in lesional skin of indicated groups. Scale bar = 50 µm. n = 6 mice. e Quantification of the dermal vascular area in the H&E-stained sections. n = 10 vision fields from 6 mice. f Quantification of extracted Evans blue dye in inflamed skin in indicated groups. n = 6 mice. g Relative mRNA expressions of inflammatory cytokines in mice tissues. n = 6 mice. The immunofluorescence staining was repeated three times independently with similar results. Mean ± SD. One-way ANOVA with Tukey’s post hoc test was performed as two-sided analyses and adjusted for multiple comparisons in the statistical analyses. ns, not significant; IMQ, imiquimod. Source data are provided as a Source Data file.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Staining, Control, Cream, Flow Cytometry, Immunofluorescence

Journal: Nature Communications

Article Title: CREB1-driven CXCR4 hi neutrophils promote skin inflammation in mouse models and human patients

doi: 10.1038/s41467-023-41484-3

Figure Lengend Snippet: This study provides insights into the development and role of CXCR4 hi neutrophils in skin inflammation. Compared to CXCR4 lo neutrophils, CXCR4 hi neutrophils exhibit increased capacity for NETs formation, phagocytosis, and degranulation, as well as higher expression of pro-inflammatory mediators, supported by activated glycolysis. CXCR4 expression on neutrophils is induced by CXCL12, IL-25, and TNF, which requires de novo mRNA and protein synthesis and intracellular protein transport, but is not stored in neutrophil granules and regulated by degranulation. CREB1 is activated by phosphorylation on Ser-133 upon stimulation, enabling interaction with its coactivator protein CBP to initiate transcription of CREB-responsive genes, thereby contributing to CXCR4 expression and CXCR4 hi neutrophils in skin inflammation. Our data further emphasize the crucial role of CXCR4 hi neutrophils in promoting vascular remodeling through lactate and MMP-9 release (B), facilitating immune cell infiltration into tissues, and increasing inflammatory responses (A). Moreover, we identify the CXCR4/CXCL12 axis as a potential therapeutic target for inflammatory skin diseases, with a focus on CREB1, CXCR4/CXCL12, or the formation of NETs. CBP, CREB-binding protein; EC, endothelial cell; MMP9, matrix metallopeptidase 9; NETs; neutrophil extracellular traps; PAD4 i, PAD4 inhibitor. Created with BioRender.com.

Article Snippet: CXCR4 hi neutrophils were also obtained by positive selection from total neutrophils using mouse CXCR4 MicroBeads (130-118-682, Miltenyi Biotec Inc.) according to the manufacturer’s protocol, as depicted below.

Techniques: Expressing, Phospho-proteomics, Binding Assay

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Fibroblast-derived CXCL12 increases vascular permeability in a 3-D microfluidic model independent of extracellular matrix contractility

doi: 10.3389/fbioe.2022.888431

Figure Lengend Snippet: Molecular characterization of fibroblasts isolated from mouse mammary glands. (A , B) Representative confocal images of cancer-associated fibroblasts (CAFs) by immunofluorescence (IF) staining of CXCL12 (green) and PDGFR-α (red for A ) and Nidogen-2 (red for B ). Nuclei (blue) were counterstained with DAPI. Scale bars are 20 µm. (C) Quantification of fluorescence intensity of PDGFR-α and Nidogen-2 per cell from confocal IF images. Values represent the mean ± standard deviation, with n = 4 technical replicates: * p ≤ 0.05, ** p ≤ 0.01, *** p < 0.001 from unpaired t -test. (D) Western blot analysis of CXCR4 from fibroblast lysates derived from mouse mammary glands. f/f: floxed control allele MMFs, ∆/∆: CXCL12-knockout MMFs, f/f; PyT: CAFs from tumor-bearing mammary glands of Cxcl12 f/f ;PyT mice. CXCR4 expression is preserved in the CXCL12 genetically ablated fibroblasts and robust in CAFs.

Article Snippet: Subsequently, primary antibodies were added to wells and incubated overnight at 4°C: CXCL12 antibody at 12.5 μg/ml concentration (R&D Systems, MAB350), PDGFR-α antibody at 1:1000 dilution (CST, 3174), and Nidogen-2 at 1:400 dilution (Abcam, ab14513).

Techniques: Isolation, Immunofluorescence, Staining, Fluorescence, Standard Deviation, Western Blot, Derivative Assay, Control, Knock-Out, Expressing

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Fibroblast-derived CXCL12 increases vascular permeability in a 3-D microfluidic model independent of extracellular matrix contractility

doi: 10.3389/fbioe.2022.888431

Figure Lengend Snippet: Compartmentalized microfluidic device for vascular permeability measurements. (A) Schematic of microtissue analogue microfluidic device. (B) Dashed boxed region in (A) depicting endothelial cell-lined microchannel laterally adjacent to a central stromal compartment containing fibroblasts embedded in a 3-D type I collagen gel. Arrows depict the endothelial-stromal compartment interface. (C) Texas Red-conjugated Dextran (70 kDa) dissolved in media served as a fluorescent tracer and was introduced into the MEEC-lined channel. The fluorescent tracer passes through the endothelial monolayer of the microvessel analogue and enters the stromal compartment by diffusion. Scale bars are 50 µm. (D) Changes in fluorescence intensity within the stromal compartment were used to calculate apparent vascular permeability of vessels in the presence of conditioned media (CM) or co-cultured with CAFs—with and without Cxcl12 gene deletion. Reported values represent the mean ± standard deviation, with n ≥ 3 biological replicates and at least 15 apertures analyzed for each condition: * p ≤ 0.05, ** p ≤ 0.01, *** p < 0.001.

Article Snippet: Subsequently, primary antibodies were added to wells and incubated overnight at 4°C: CXCL12 antibody at 12.5 μg/ml concentration (R&D Systems, MAB350), PDGFR-α antibody at 1:1000 dilution (CST, 3174), and Nidogen-2 at 1:400 dilution (Abcam, ab14513).

Techniques: Permeability, Diffusion-based Assay, Fluorescence, Cell Culture, Standard Deviation

Journal: iScience

Article Title: Blockade of TSP-1/CD47 signal axis promotes donor hematopoietic engraftment by improving SEC/MK niche function

doi: 10.1016/j.isci.2025.111952

Figure Lengend Snippet: CXCL12 expression pattern at 24 h post 9 Gy TBI in three genetic mice (A) Representative immunofluorescent images of CXCL12 distribution (green channel) at sinusoidal/megakaryocyte niches in cortical region or mid-plane of WT (top panel), Cd47 −/− (middle panel), and Thbs1 −/− (bottom panel) mice at 24 h post-TBI. White arrows highlight megakaryocytes, and white arrowheads indicate thrombi in the sinusoidal space. Scale bar = 50 μm. For corresponding whole mount immunofluorescent images of the bone marrow sections for all three groups, please refer to Figure S5 . (B) Average CXCL12 expression levels were quantified using ImageJ (left), with each symbol representing a single Z montage image. CXCL12 expression in megakaryocytes (middle), or in thrombi (right) was quantified using Imaris, with data collected from 10 to 12 fields at 20 × objective from 3 mice. (C) CXCL12 concentration in BM supernatant in three genetic mice at 24 h post 9 Gy TBI examined by Elisa ( n = 4). Data were presented as mean ± S.D., ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001, determined by one-way ANOVA with Tukey’s multiple comparison test (B and C).

Article Snippet: Bone marrow supernatant was diluted 10-fold with sample diluent, and the concentration of CXCL12 was measured using an ELISA kit (Elabscience, E-EL-M3046) according to the manufacturer’s instructions.

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: iScience

Article Title: Blockade of TSP-1/CD47 signal axis promotes donor hematopoietic engraftment by improving SEC/MK niche function

doi: 10.1016/j.isci.2025.111952

Figure Lengend Snippet:

Article Snippet: Bone marrow supernatant was diluted 10-fold with sample diluent, and the concentration of CXCL12 was measured using an ELISA kit (Elabscience, E-EL-M3046) according to the manufacturer’s instructions.

Techniques: Recombinant, Staining, Cell Isolation, TUNEL Assay, In Situ, Derivative Assay, Enzyme-linked Immunosorbent Assay, Bicinchoninic Acid Protein Assay, Software

Journal: eLife

Article Title: Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice

doi: 10.7554/elife.92644

Figure Lengend Snippet: Figure 8. Coculture of AAV transduced SC-derived myotubes with rat spinal motor neurons in compartmentalized microfluidic chambers. (A) Schematic representation of XonaChip spatial configuration and coculture experiment timeline. NBPM: Neurobasal Plus Medium; RSMNs: rat spinal motor neurons. (B) Representative composite images of RSMNs cocultured with: 1. WT HL SC-derived myotubes transduced with AAV-CMV-eGFP; 2. G93A HL SC-derived myotubes transduced with AAV- CMV-eGFP; 3. G93A HL SC-derived myotubes transduced with AAV-CMV-Cxcl12-IRES-eGFP; 4. G93A EOM

Article Snippet: Peptide, recombinant protein Collagenase II Worthington Cat # LS004176 Final concentration 0.26% Peptide, recombinant protein Dispase II Sigma Aldrich Cat # D46931G Final concentration 0.24% Peptide, recombinant protein Hyaluronidase Worthington Cat # LS002592 Final concentration 0.16% Peptide, recombinant protein DNase I Worthington Cat # LS002139 Final concentration 0.04% Antibody Anti- mouse Vcam1- PE (Rat monoclonal IgG2a) BioLegend Clone 429 (MVCAM.A) Cat # 105713 600 ng/106 cells for FACS isolation Antibody Anti- mouse CD31- APC (Rat monoclonal IgG2a) BioLegend Clone 390 Cat #102409 400 ng/106 cells for FACS isolation Antibody Anti- mouse CD45- APC (Rat monoclonal IgG2b) BioLegend Clone 30- F11 Cat # 103111 400 ng/106 cells for FACS isolation Antibody Anti- mouse Sca- 1- APC (Rat monoclonal IgG2a) BioLegend Clone D7 Cat # 108111 200 ng/106 cells for FACS isolation Antibody Anti- chicken Pax7 (Mouse monoclonal IgG1) Santa Cruz Biotechnology Cat # sc- 81648 IF (1:100) Antibody Anti- mouse MyoD (Mouse monoclonal IgG2b) Santa Cruz Biotechnology Cat # sc- 377460 IF (1:100) Antibody Anti- mouse Ki- 67 (Rabbit monoclonal IgG) Cell Signaling Technology Cat # 12202 S IF (1:300) Antibody Anti- chicken MHC (Mouse monoclonal IgG2b) Developmental Studies Hybridoma Bank Cat # MF20 IF (1:200) Antibody Anti- rat NF- M (Mouse monoclonal IgG1) Developmental Studies Hybridoma Bank Cat # 2H3 IF (1:300) Antibody Anti- GFP (Mouse monoclonal IgG2a) Novus Clone 4B10B2 Cat # NBP222111AF488 IF (1:250) Antibody Anti- rat Synaptophysin (Rabbit polyclonal IgG) Thermo Fisher Scientific PA11043 IF (1:400) Antibody Anti- human Cxcl12 (Mouse monoclonal IgG1) R&D systems Clone 79018 Cat # MAB350 IF (1:200) Other AF 488 conjugated α-Bungarotoxin Thermo Fisher Scientific Cat # B13422 IF (1:1000) Continued Continued on next page Li et al. eLife 2023;12:RP92644.

Techniques: Derivative Assay, Transduction

Journal: eLife

Article Title: Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice

doi: 10.7554/elife.92644

Figure Lengend Snippet: Figure 9. Coculture of AAV transduced SC-derived myotubes with rat spinal motor neurons within the same compartment. (A) Representative composite images of RSMNs cocultured with: 1. WT HL SC-derived myotubes transduced with AAV-CMV-eGFP; 2. G93A HL SC-derived myotubes transduced with AAV-CMV-eGFP; 3. G93A HL SC-derived myotubes transduced with AAV-CMV- Cxcl12-IRES-eGFP; 4. G93A EOM SC-derived myotubes transduced with AAV-CMV-eGFP. The timeline was the same as that in Figure 8 but RSMNs were seeded on top of myotubes. Boxed regions are

Article Snippet: Peptide, recombinant protein Collagenase II Worthington Cat # LS004176 Final concentration 0.26% Peptide, recombinant protein Dispase II Sigma Aldrich Cat # D46931G Final concentration 0.24% Peptide, recombinant protein Hyaluronidase Worthington Cat # LS002592 Final concentration 0.16% Peptide, recombinant protein DNase I Worthington Cat # LS002139 Final concentration 0.04% Antibody Anti- mouse Vcam1- PE (Rat monoclonal IgG2a) BioLegend Clone 429 (MVCAM.A) Cat # 105713 600 ng/106 cells for FACS isolation Antibody Anti- mouse CD31- APC (Rat monoclonal IgG2a) BioLegend Clone 390 Cat #102409 400 ng/106 cells for FACS isolation Antibody Anti- mouse CD45- APC (Rat monoclonal IgG2b) BioLegend Clone 30- F11 Cat # 103111 400 ng/106 cells for FACS isolation Antibody Anti- mouse Sca- 1- APC (Rat monoclonal IgG2a) BioLegend Clone D7 Cat # 108111 200 ng/106 cells for FACS isolation Antibody Anti- chicken Pax7 (Mouse monoclonal IgG1) Santa Cruz Biotechnology Cat # sc- 81648 IF (1:100) Antibody Anti- mouse MyoD (Mouse monoclonal IgG2b) Santa Cruz Biotechnology Cat # sc- 377460 IF (1:100) Antibody Anti- mouse Ki- 67 (Rabbit monoclonal IgG) Cell Signaling Technology Cat # 12202 S IF (1:300) Antibody Anti- chicken MHC (Mouse monoclonal IgG2b) Developmental Studies Hybridoma Bank Cat # MF20 IF (1:200) Antibody Anti- rat NF- M (Mouse monoclonal IgG1) Developmental Studies Hybridoma Bank Cat # 2H3 IF (1:300) Antibody Anti- GFP (Mouse monoclonal IgG2a) Novus Clone 4B10B2 Cat # NBP222111AF488 IF (1:250) Antibody Anti- rat Synaptophysin (Rabbit polyclonal IgG) Thermo Fisher Scientific PA11043 IF (1:400) Antibody Anti- human Cxcl12 (Mouse monoclonal IgG1) R&D systems Clone 79018 Cat # MAB350 IF (1:200) Other AF 488 conjugated α-Bungarotoxin Thermo Fisher Scientific Cat # B13422 IF (1:1000) Continued Continued on next page Li et al. eLife 2023;12:RP92644.

Techniques: Derivative Assay, Transduction

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: Pretreatment with CXCL12 promotes transition from G1 to S phase during growth factor deprivation (GFD). ( A-C ) Flow cytometry analysis shows the cell cycle phase of P13.5 mouse neural progenitor cells which were pretreated with CXCL12 for 2 hours and then treated without growth factors for 0 to 12 hours. ( D ) Results are presented as the mean ± SEM of three independent experiments with NPC from three mouse donors. *** P <0.001, significantly different from control group, ## P <0.01, ### P <0.001, significantly different from GFD group.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Flow Cytometry, Control

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: Real time PCR and Western blot analysis of CyclinD and β-catenin following CXCL12 pretreatment. ( A ) mNPCs pretreated with CXCL12 at different concentrations (0,10,50,100 ng/ml) for 2 hours and then treated without growth factors for 24 hours. Values are means ± SEM (n=3). ( B ) mNPC pretreated with CXCL12 at 0, 50, 100, 250 ng/ml for 2 hours following 24 hour starvation. Values are means ± SEM (n=3), * P <0.05, ** P <0.01, significantly different from the GFD group. ( C ) mNPCs were pretreated with CXCL12 for 2 hours and then treated without growth factors for 24 hours. ( D ) Levels of β-catenin were normalized as a ratio of β-catenin to actin after densimetrical quantification of panel C and shown as fold change relative to GFD group. Results are expressed as the mean ± SEM of triplicate samples and are representative of 3 independent experiments. * P <0.05, compared with GFD.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Real-time Polymerase Chain Reaction, Western Blot

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: Proliferation effect of CXCL12 on mouse NPC by Ki67 staining. CXCL12-mediated proliferation of mouse NPC is determined by Ki67 staining after pretreatment of increasing concentrations of CXCL12 stimulation for 2 hours followed by the deprivation of growth factors for 24 hours. Cell proliferation was assessed by immuno-staining for Ki67 positive in mNPC in GFD group ( A,B,C ) and with CXCL12 pretreatment group ( D,E,F ). The percentage of Ki67-positive proliferating mNPCs were determined for each treatment with different concentrations of CXCL12 by counting positive cells per microscopic field in 30 pictures per condition ( G,H ). Results are presented as the mean ± SEM of three independent experiments with NPC from three mouse donors. * P <0.05, ** P <0.01, in comparison to GFD group, scale bar=100 μm.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Staining, Immunostaining, Comparison

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: CXCL12 pretreatment protected mNPC against apoptosis induced by the deprivation of growth factors. mNPCs were pretreated with CXCL12 for 2 hours and then deprived of growth factors for 24 hours. mNPCs apoptosis was determined by TUNEL assay ( A-D ) or PARP cleavage assay ( F ). ( A-D ) Representative pictures from TUNEL assay are shown. ( E ) Quantification data of TUNEL assay were determined as a percentage of TUNEL positive cells against the total cell number. ( F ) Cleaved PARP protein levels were determined by Western blotting. ( G ) Levels of cleaved PARP were normalized as a ratio of cleaved PARP to actin after densimetrical quantification of panel F and shown as fold change relative to control. Results are expressed as the mean ± SEM of triplicate samples and are representative of 3 independent experiments. ## P <0.01 compared with control; * P <0.05, ** P <0.01 compared with growth factors deprivation group without CXCL12 pretreatment. Scale bar=100 μm.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: TUNEL Assay, Cleavage Assay, Western Blot, Control

Journal: Current Molecular Medicine

Article Title: CXCR7 Participates in CXCL12-mediated Cell Cycle and Proliferation Regulation in Mouse Neural Progenitor Cells

doi: 10.2174/1566524016666160829153453

Figure Lengend Snippet: CXCR7 is required for the proliferation function of CXCL12 in mNPCs. ( A-D ) immuno-staining for DAPI in CXCR4 knock out and CXCR7 knock out mNPCs in the starvation group ( A,C ) and with the CXCL12 pretreatment group ( B,D ). The total cell numbers were determined for each treatment with CXCL12 by counting positive cells per microscopic field in 30 pictures per condition ( E ). Results are presented as the mean ± SEM of three independent experiments with NPC from three mouse donors. ns>0.05 in comparison to starvation. Scale bar=100 μm.

Article Snippet: Recombinant mouse CXCL12 was obtained from R&D Systems (Minneapolis, MN, www.rndsystems.com ).

Techniques: Immunostaining, Knock-Out, Comparison