Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Expression of NOD2 is elevated in the lung tissue of patients with COPD The expression of NOD2 was evaluated in lung tissue samples from healthy subjects and patients with COPD. ( A ) Schematic diagram of the experimental workflow for analyzing human lung tissue. ( B ) Quantitative real-time PCR (qRT-PCR) analysis of NOD2 mRNA. ( C ) Representative images of immunohistochemical (IHC) staining for NOD2 protein. ( D ) Quantification of IHC staining. ( E ) Representative images of fluorescence in situ hybridization (FISH) for NOD2 mRNA (green), with nuclei counterstained with DAPI (blue). ( F ) Quantification of FISH signal. ( G ) Representative multicolor immunofluorescence images showing CD66b-positive neutrophils (pink) and NOD2 (green), with nuclei counterstained with DAPI (blue). Enlarged views of the boxed regions are shown on the right. Scale bars: 20 μm. ( H ) Immunoblotting analysis and quantification of NOD2 protein levels in differentiated human HL-60-derived neutrophil-like cells and primary mouse neutrophils following CSE stimulation for 0, 2, 4, or 8 h, with untreated cells serving as the 0-h controls. ( I) qRT-PCR analysis of NOD2 mRNA expression in the corresponding human and mouse neutrophil models. Data are presented as mean ± SD. Data in B, D, and F were analyzed using an unpaired two-tailed Student’s t-test; data in H and I were analyzed separately for each cell model using one-way ANOVA with Tukey’s test. ** p < 0.01 versus the corresponding healthy-subject or CSE 0-h control group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Expressing, Tissue, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Analysis, Immunohistochemical staining, Immunohistochemistry, Fluorescence, In Situ Hybridization, Immunofluorescence, Western Blot, Derivative Assay, Two Tailed Test, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: NOD2 deficiency ameliorates cigarette smoke-induced lung inflammation and injury in vivo The functional role of NOD2 was evaluated in a cigarette smoke (CS)-induced mouse model of COPD using wild-type (wt) and NOD2-knockout (NOD2-/-) mice, with lentiviral-mediated NOD2 re-expression (NOD2-OE) in NOD2-/- COPD mice. ( A ) Schematic of the in vivo experimental design. ( B ) qRT-PCR analysis of NOD2 mRNA expression in lung tissue. ( C ) Representative images of hematoxylin and eosin (H&E) staining of lung tissue sections. Scale bars: 200 μm in the upper panels and 100 μm in the enlarged lower panels. ( D ) Representative images of Masson’s trichrome staining for collagen deposition in lung tissue sections. Scale bar: 100 μm. ( E ) Quantification of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF), and ELISA measurements of TNF-α, IL-6, and CXCL1 levels in BALF supernatant. ( F ) Representative images of IHC staining for E-cadherin and N-cadherin in lung tissue. Scale bar: 50 μm. ( G ) Quantification of the average optical density (AOD) for E-cadherin and N-cadherin IHC staining. ( H ) qRT-PCR analysis of senescence-associated secretory phenotype (SASP) factors (CSF3, CCL3, and IL1α) in lung tissue. Data are presented as mean ± SD. Data were analyzed using one-way ANOVA followed by Tukey’s test. * p < 0.05 and ** p < 0.01 Control vs. wt-COPD groups; # p < 0.05 and ## p < 0.01 wt-COPD vs. NOD2 −/− -COPD groups; & p < 0.05 and && p < 0.01 NOD2 −/− -COPD + vector vs. NOD2 −/− -COPD + NOD2-OE groups

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: In Vivo, Functional Assay, Knock-Out, Expressing, Quantitative RT-PCR, Analysis, Tissue, Staining, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Control, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: NOD2 activation induced excessive mitophagy in the lungs of CS-exposed mice The role of NOD2 in regulating mitophagy was assessed in lung tissue and primary neutrophils isolated from mice in the CS-induced COPD model. ( A ) Representative transmission electron microscopy (TEM) images showing mitochondrial ultrastructure and mitophagosome-like structures in lung tissue. Insets show magnified views of mitochondrial morphology and mitochondria-containing autophagosomes. Scale bars: 500 nm. ( B ) Representative TEM images showing mitochondrial ultrastructure and mitophagosome-like structures in primary neutrophils isolated from the corresponding mouse groups. Insets show magnified views of mitochondrial morphology and mitochondria-containing autophagosomes. Scale bars: 500 nm. ( C ) Immunoblotting analysis of mitophagy-related proteins (Beclin1, LC3I/II, PINK1, Parkin, NIX, and BNIP3) in whole-lung tissue lysates. Data are presented as mean ± SD. Data in C were analyzed using one-way ANOVA followed by Tukey’s test. ** p < 0.01 Control vs. wt -COPD groups; and ## p < 0.01 wt -COPD vs. NOD2 −/− -COPD groups; & p < 0.05 and && p < 0.01 NOD2 −/− -COPD + vector vs. NOD2 −/− -COPD + NOD2-OE groups

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Activation Assay, Tissue, Isolation, Transmission Assay, Electron Microscopy, Morphology, Western Blot, Analysis, Control, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: NOD2 promotes Neutrophil Extracellular Trap (NET) formation in the CS-induced COPD mouse model The association between NOD2, autophagy, and Neutrophil Extracellular Trap (NET) formation was investigated in the lung tissue of mice from the CS-induced COPD model. ( A) Representative immunofluorescence images showing co-localization of the neutrophil marker Ly6G (green) and the autophagy marker LC3B (red), with nuclei counterstained with DAPI (blue). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. ( B ) Representative immunofluorescence images showing staining for the NET markers citrullinated histone H3 (CitH3, green) and myeloperoxidase (MPO, red), with nuclei counterstained with DAPI (blue). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. ( C ) Quantification of the NET biomarkers CitH3-DNA and MPO-DNA complexes in serum and lung tissue homogenates by ELISA. ( D ) Immunoblotting analysis of the neutrophil marker Ly6G and the NET marker CitH3 in whole-lung tissue lysates. ( E ) Representative immunofluorescence images showing colocalization of the neutrophil marker Ly6G (red), the NET marker CitH3 (green), and NOD2 (pink) in lung tissue, with nuclei counterstained with DAPI (blue). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. Data are presented as mean ± SD. C and D were analyzed using one-way ANOVA followed by Tukey’s test. ** p < 0.01 Control vs. wt -COPD groups; and ## p < 0.01 wt -COPD vs. NOD2 −/− -COPD groups; && p < 0.01 NOD2 −/− -COPD + vector vs. NOD2 −/− -COPD + NOD2-OE groups

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Tissue, Immunofluorescence, Marker, Staining, Enzyme-linked Immunosorbent Assay, Western Blot, Analysis, Control, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Neutrophil depletion minimizes genotype-dependent differences in CS-induced lung injury, inflammation, fibrosis, and NET formation The dependence of NOD2-associated COPD pathology on neutrophils was evaluated by depleting neutrophils in CS-exposed wild-type (wt) and NOD2 −/− mice. Mice were randomly assigned to the wt -COPD + IgG, wt -COPD + Anti-Ly6G, NOD2 −/− -COPD + IgG, and NOD2 −/− -COPD + Anti-Ly6G groups ( n = 6 per group). ( A ) Schematic of the experimental design. Mice were exposed to CS for 12 weeks and received 200 µg of anti-Ly6G antibody or an equivalent dose of isotype IgG intraperitoneally three times per week from week 2 until the end of the experiment. ( B ) Representative flow-cytometry plots and quantification of CD11b + Ly6G + neutrophils in lung tissue and peripheral blood. ( C ) Representative H&E-stained images of lung tissue sections. Scale bars: 200 μm in the upper panels and 100 μm in the enlarged lower panels. ( D ) Representative images of Masson’s trichrome staining for pulmonary collagen deposition. Scale bars: 100 μm. ( E ) Quantification of total cells, neutrophils, and macrophages in bronchoalveolar lavage fluid (BALF), together with ELISA measurements of TNF-α, IL-6, and CXCL1 levels in BALF supernatants. ( F ) Quantification of CitH3-DNA and MPO-DNA complexes in serum and lung tissue homogenates by ELISA. Data are presented as mean ± SD. Data were analyzed using two-way ANOVA followed by Tukey’s test. ** p < 0.01 vs. the wt -COPD + IgG group; ## p < 0.01 vs. the wt-COPD + Anti-Ly6G group; & p < 0.05, & p < 0.01 vs. the NOD2 −/− -COPD + IgG group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Pathology, Flow Cytometry, Tissue, Staining, Enzyme-linked Immunosorbent Assay

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Knockdown of NOD2 inhibits CSE-induced NET formation, ROS production, and mitophagy in human neutrophils The role of NOD2 in cigarette smoke extract (CSE)-induced Neutrophil Extracellular Trap (NET) formation, reactive oxygen species (ROS) accumulation, and lysosome–mitochondrion spatial relationships were evaluated in differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the in vitro experimental design and treatment groups. ( B ) Representative DAPI-stained images of extracellular DNA structures and quantification of NET formation based on DAPI-stained nuclear morphology. Scale bars: 20 μm. ( C ) Extracellular DNA levels in cell culture supernatants determined using a PicoGreen dsDNA assay. ( D ) Myeloperoxidase (MPO) levels in cell culture supernatants determined by ELISA. ( E ) Neutrophil elastase (NE) levels in cell culture supernatants determined by ELISA. ( F ) Representative flow-cytometry histograms and quantitative analysis of intracellular ROS levels measured using DCFH-DA staining. ( G ) Representative immunofluorescence images showing the spatial relationship between LAMP1-positive lysosomes (green) and TOM20-positive mitochondria (red), with nuclei counterstained with DAPI (blue). The colocation efficients for LAMP1 and TOM20 was determined by ImageJ and shown on the right. Scale bars: 20 μm. Data are presented as mean ± SD. Quantitative data were analyzed using one-way ANOVA followed by Tukey’s test. ** p < 0.01 vs. Control group; ## p < 0.01 vs. CSE + sh-NC group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Knockdown, Derivative Assay, In Vitro, Staining, Morphology, Cell Culture, Picogreen Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Analysis, Immunofluorescence, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: ZDHHC5-mediated S-palmitoylation is essential for NOD2 stability and signaling The association of ZDHHC5 with NOD2 and the contribution of NOD2 S-palmitoylation to its stability, localization, and downstream signaling were investigated in differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the proposed mechanism by which ZDHHC5-mediated S-palmitoylation promotes NOD2 membrane localization. ( B ) Representative immunofluorescence images showing NOD2 (red) in cells with or without CSE stimulation. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm. ( C ) Acyl-biotin exchange (ABE) assay evaluating Flag-NOD2 S-palmitoylation in cells with or without CSE stimulation. HAM, hydroxylamine. ( D ) Immunoblotting analysis and quantification of NOD2 protein levels following treatment with increasing concentrations of the palmitoylation inhibitor 2-bromopalmitate (2BP) or with 100 µM 2BP for the indicated durations. ( E ) Representative immunofluorescence images showing NOD2 (red) in cells treated with vehicle or 2BP. Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm. ( F ) Co-immunoprecipitation analysis of the association between endogenous ZDHHC5 and NOD2. ( G ) Representative immunofluorescence images showing ZDHHC5 (green) and NOD2 (red), with nuclei counterstained with DAPI (blue). Scale bars: 20 μm. ( H ) Schematic of human NOD2 showing its functional domains and the positions of the C395S and C1033S mutations. ( I ) Immunoblotting analysis of wild-type (WT) and mutant Flag-NOD2 in total-cell lysates and membrane fractions during a cycloheximide (CHX) chase, with quantification of the relative NOD2 protein remaining shown on the right. GAPDH and Na + /K + -ATPase were used as loading controls for the total-cell lysates and membrane fractions, respectively. ( J ) ABE assay comparing the S-palmitoylation levels of WT Flag-NOD2, the C395S and C1033S single mutants, and the C395S/C1033S double mutant. ( K ) Immunoblotting analysis and quantification of p65 and p38 phosphorylation in cells expressing wt Flag-NOD2 or the C395S/C1033S double mutant following CSE stimulation. Data are presented as mean ± SD. Data in D and K were analyzed using one-way ANOVA followed by Tukey’s test. Data in I were analyzed using two-way ANOVA followed by Tukey’s test. ** p < 0.01 versus the corresponding 0 µM, 0 h, or wt control group; ## p < 0.01 versus the CSE + Flag-NOD2 wt group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Stability, Derivative Assay, Membrane, Immunofluorescence, Western Blot, Analysis, Co-Immunoprecipitation Assay, Functional Assay, Mutagenesis, Phospho-proteomics, Expressing, Control

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: ZDHHC5 overexpression partially counteracts the effects of NOD2 knockdown in human neutrophil-like cells The ability of ZDHHC5 overexpression to counteract the effects of NOD2 knockdown was evaluated in CSE-stimulated, differentiated HL-60-derived neutrophil-like cells. ( A ) Schematic of the in vitro experimental design and treatment groups. ( B ) Representative DAPI-stained images of extracellular DNA structures and quantification of NET formation based on DAPI-stained nuclear morphology. Scale bars: 20 μm. ( C ) Extracellular DNA levels in cell culture supernatants determined using a PicoGreen dsDNA assay. ( D ) MPO levels in cell culture supernatants determined by ELISA. ( E ) NE levels in cell culture supernatants determined by ELISA. ( F ) Representative flow cytometry histograms and quantitative analysis of intracellular ROS levels measured using DCFH-DA staining. ( G ) Representative immunofluorescence images showing LAMP1-positive lysosomes (green) and TOM20-positive mitochondria (red), with nuclei counterstained with DAPI (blue). The colocalization coefficients for LAMP1 and TOM20 were determined by ImageJ and shown on the right. Scale bars: 20 μm. Data are presented as mean ± SD. Data were analyzed using two-way ANOVA followed Tukey’s test. ** p < 0.01 versus the CSE + sh-NC + Vector group; ## p < 0.01 versus the CSE + sh-NOD2 + ZDHHC5 group

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Over Expression, Knockdown, Derivative Assay, In Vitro, Staining, Morphology, Cell Culture, Picogreen Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Analysis, Immunofluorescence, Plasmid Preparation

Journal: Biomarker Research

Article Title: ZDHHC5-mediated S-palmitoylation of NOD2 drives neutrophil extracellular trap formation via mitophagy to exacerbate inflammation in COPD

doi: 10.1186/s40364-026-01001-2

Figure Lengend Snippet: Proposed model linking ZDHHC5-associated NOD2 S-palmitoylation to mitophagy and NET formation in COPD. CS exposure increases NOD2 and ZDHHC5 expression in pulmonary neutrophils. ZDHHC5-mediated NOD2 S-palmitoylation supports NOD2 stability and membrane localization, promoting NF-κB/MAPK signaling and mitophagy, intracellular ROS accumulation, and NET formation. Additional NOD2-independent effects of ZDHHC5 may also contribute to COPD-associated inflammation

Article Snippet: Primary neutrophils were isolated from BALF of the indicated mouse groups using a neutrophil isolation kit (Miltenyi Biotec).

Techniques: Expressing, Stability, Membrane

Journal: bioRxiv

Article Title: Radiotherapy-induced vascular dysfunction alters neutrophil phenotype to promote tumor cell colonization of mammary adipose tissue

doi: 10.64898/2026.10.03.756445

Figure Lengend Snippet: a. Experimental timeline for CD8 + T cell redution experiments. Female BALB/c mice were inoculated with 4T1 luc cells and treated with 500µg of CD8 or IgG control antibodies at the time of inoculation and every 5 days therafter. When tumors were 100mm 3 , the contralateral MFP was irradiate to 20 Gy and MFPs were collected for downstream analysis at 10 days post-RT. Representative images (b) and quantification (c) of Ly6G + cells in MFPs of BALB/c mice. n=5 mice per condition. **p<0.01 and ***p<0.001 by ANOVA with Sidak’s test. d. Quantification of CD45 + CD11b + Ly6C mid Ly6G + SSC hi neutrophils in MFPs at 10d post-RT analyzed via flow cytometry. n=8-12 mice per condition. p-values determined by ANOVA with Sidak’s test. e. Experimental timeline for Nu/Nu experiments with details as noted above except antibody treatments. f. Quantification of CD45 + CD11b + Ly6C mid Ly6G + neutrophils in MFPs of Nu/Nu mice at 10d post-RT analyzed via flow cytometry. n=5 mice per condition. p-value determined by t-test. (g) Significantly enriched pathways related to tissue remodeling in irradiated CD8 mice determined by Reactome analysis of MFP bulk RNA-seq data. n=5 mice per condition. h. Representative images (b) and quantification (c) of percent CD31 + area in MFPs of BALB/c mice. n=5 mice per condition. *p<0.05 and ***p<0.001 by ANOVA with Sidak’s test. j. Experimental timeline for neutrophil depletion model. Nu/Nu mice were inoculated and treated as in (e) with additional i.p. injections with Ly6G or IgG control antibodies at the time of inoculation and every other day thereafter. k. Fold change of lucb expression relative to Actb in 20 Gy vs. 0 Gy contralateral MFPs via RT-qPCR. n=8-12 mice per condition. p-value determined by Mann-Whitney test.

Article Snippet: Magnetic-activated cell sorting (MACS) was then performed using a mouse neutrophil isolation kit (Miltenyi) to negatively select for Ly6G + cells.

Techniques: Control, Irradiation, Analysis, Flow Cytometry, Tissue, RNA Sequencing, Expressing, Quantitative RT-PCR, MANN-WHITNEY, Protein-Protein interactions, Significance Assay

Journal: bioRxiv

Article Title: Radiotherapy-induced vascular dysfunction alters neutrophil phenotype to promote tumor cell colonization of mammary adipose tissue

doi: 10.64898/2026.10.03.756445

Figure Lengend Snippet: a. Representative images of CD31-VEGF costaining of MFPs in Nu/Nu neutrophil depletion model. Scale bars are 50µm. Coresponding quantification of CD31 + vessels per mm 2 (b) and integrated density of VEGF localized with CD31 + vessels (c). n=4-5 mice per condition. *p<0.05 and **p<0.01 determined by two-way ANOVA with Sidak’s test. Quantification (d) and (e) representative images of F4/80 + cells per mm 2 in MFPs from Nu/Nu neutrophil depletion model. n=3-5 mice per condition. ***p<0.001 determined by two-way ANOVA with Sidak’s test. f. Gene expression of select genes related to a pathologically-activated PMN-MDSC phenotype from bulk RNA-seq analysis of MFPs of IgG and CD8 mice in 20 vs. 0 Gy MFPs. n=5 mice per condition. g. Representative images of neutrophil extracellular traps (NETs) via localization of DAPI, MPO, and CitH3 in MFPs of Nu/Nu mice bearing contralateral 4T1 luc tumors. Arrows indicate localization of all three stains. Quantification (h) and representative images (i) of S100A9 + cells in MFPs from Nu/Nu-4T1 luc model. n=2-5 mice per condition j. Flow cytometry analysis of expression of key PMN-MDSC markers shown as percent of neutrophils. Neutrophils were defined as live CD45 + Ly6C mid Ly6G + cells in MFP, spleen, blood, and bone marrow from Nu/Nu 4T1 luc model. n=5 mice per condition. **p<0.01 and ***p<0.001 by two-way ANOVA with organ as the repeated measures factor followed by Sidak’s test. All data show mean±SD. All error bars are 50µm.

Article Snippet: Magnetic-activated cell sorting (MACS) was then performed using a mouse neutrophil isolation kit (Miltenyi) to negatively select for Ly6G + cells.

Techniques: Gene Expression, RNA Sequencing, Analysis, Flow Cytometry, Expressing

Journal: bioRxiv

Article Title: Radiotherapy-induced vascular dysfunction alters neutrophil phenotype to promote tumor cell colonization of mammary adipose tissue

doi: 10.64898/2026.10.03.756445

Figure Lengend Snippet: a. Representative images of CD31/p21 costaining of MFPs from Nu/Nu model at 10 days post-RT. Arrows indicate examples of vessels (CD31) expressing p21. b. Experimental timeline for in vitro endothelial cell experiments. C166 ECs were irradiated to 10 Gy and cultured for 2, 5, or 7 days thereafter before analysis for hallmarks of senescence. Quantification (c) and representative images (d) of EdU incorporation in ECs post-RT. n=3 independent replicates per condition. e. Quantification of percent positive p21 staining in and representative images of ECs post-RT. n=3 independent replicates per condition. ***p<0.001 determined by ANOVA with Sidak’s. f. Representative images of SA-β-gal activity in ECs at 7 days post-RT. g. Cytokine profiling of EC conditioned media at 2 and 7 days post-RT. n=3 independent replicates per condition. Fold change calculated with respect to the average cytokine concentration of the 0 Gy sample at each corresponding timepoint. P-value determined by ANOVA with Sidak’s. h. Significantly upregulated SASP cytokines in MFP homogenate from BALB/c mice at 10 days post-RT. n=5 mice per condition. *p<0.05 and ***p<0.001 determined by ANOVA with Sidak’s. i. Experimental timeline of BMDN isolation and subsequent incubation in EC conditioned media. j. Gene expression of PMN-MDSC-related genes relative to Actb in BMDNs after incubation in EC CM. Error bars show SD. Quantification via flow cytometry of CD84 expression (k) and NETosis induction (SYTOX+ cells) (l) in Ly6G + Ly6C mid BMDNs after incubation in EC conditioned media. n=5 and 2 independent experiments for NETosis and CD84 analysis, respectively. For CD84 analysis, p-values determined by paired t-test. m. Representative images of DAPI-stained irradiated (10 Gy) and control EC monolayers after neutrophil co-culture. Arrows show examples of EC (yellow) and neutrophil (blue) nuclei and activated neutrophils (red). All scale bars are 50 μm.

Article Snippet: Magnetic-activated cell sorting (MACS) was then performed using a mouse neutrophil isolation kit (Miltenyi) to negatively select for Ly6G + cells.

Techniques: Expressing, In Vitro, Irradiation, Cell Culture, Analysis, EdU Assay, Staining, Activity Assay, Profiling, Concentration Assay, Isolation, Incubation, Gene Expression, Flow Cytometry, Control, Co-culture Assay, Magnetic Cell Separation

Journal: bioRxiv

Article Title: Radiotherapy-induced vascular dysfunction alters neutrophil phenotype to promote tumor cell colonization of mammary adipose tissue

doi: 10.64898/2026.10.03.756445

Figure Lengend Snippet: a. Experimental timeline of neutrophil-EC co-culture experiments. ECs were irradiated to 10 Gy. At 24h prior to each timepoint of interest, co-cultures were initiated and cells were cultured for 48h thereafter before downstream gene and protein expression analysis. b. Western blot analysis of phosphorylated-p65 expression in ECs with and without neutrophil co-cultures. Protein expression was normalized to a loading control and fold change relative to each 0 Gy culture condition was calculated. n=3-5 independent experiments per condition. *p<0.05 and **p<0.01 by ANOVA with Sidak’s test. Gene expression analysis of genes related to NF-κB signaling (c) and vascular dysfunction/inflammation (d) in ECs with and without neutrophil co-culture. e. Experimental timeline for FITC-dextran permeability assays with EC Transwell monolayer model. Inserts were irradiated and cultured for 24h before performing neutrophil migration and subsequent FITC-dextran permeability assay. f. Quantification of apparent permeability of endothelial monolayers post-RT and post-neutrophil migration. n=5-12 independent experiments. ***p<0.001 by ANOVA with Sidak’s. g. Gene expression analysis to confirm knockdown of Rela and inhibition of downstream NF-κB signaling in irradiated si Rela and scramble control ECs up to 7 days post-RT. h. FITC-dextran permeability of si Rela and scramble control EC monlayers after RT and neutrophil migration. n=2 independent experiments. i. Gene expression analysis of si Rela and scramble control ECs post-RT.

Article Snippet: Magnetic-activated cell sorting (MACS) was then performed using a mouse neutrophil isolation kit (Miltenyi) to negatively select for Ly6G + cells.

Techniques: Co-culture Assay, Irradiation, Co-Cultures, Cell Culture, Expressing, Analysis, Western Blot, Control, Gene Expression, Permeability, Migration, FITC-Dextran Permeability Assay, Knockdown, Inhibition, Co-Culture

Journal: bioRxiv

Article Title: Radiotherapy-induced vascular dysfunction alters neutrophil phenotype to promote tumor cell colonization of mammary adipose tissue

doi: 10.64898/2026.10.03.756445

Figure Lengend Snippet: RT induces an NF-κB-dependent pro-inflammatory secretome from senescent ECs causes pathological neutrophil activation and subsequent amplification of NF-κB signaling, perpetuating a pathological neutrophil-EC feedback loop. This interaction may lead to tumor cell colonization in T lymphocyte-deficient mice, wherein RT induces a pathological neutrophil phenotype that coincides with neutrophil dependent vascular remodeling and tumor cell colonization.

Article Snippet: Magnetic-activated cell sorting (MACS) was then performed using a mouse neutrophil isolation kit (Miltenyi) to negatively select for Ly6G + cells.

Techniques: Activation Assay, Amplification

Journal: bioRxiv

Article Title: Radiotherapy-induced vascular dysfunction alters neutrophil phenotype to promote tumor cell colonization of mammary adipose tissue

doi: 10.64898/2026.10.03.756445

Figure Lengend Snippet: a. Flow cytometry analysis of CD45 + CD11b + Ly6C mid Ly6G + cells in Nu/Nu-4T1 luc neutorphil depletion model without RT. n=6-9 mice per condition. ***p<0.001 by Mann-Whitney test. b. Mouse metrics over time in Nu/Nu-4T1 luc neutrophil depletion model with RT. n=5 mice per condition. Data show mean±SD.

Article Snippet: Magnetic-activated cell sorting (MACS) was then performed using a mouse neutrophil isolation kit (Miltenyi) to negatively select for Ly6G + cells.

Techniques: Flow Cytometry, Analysis, MANN-WHITNEY