Journal: iScience

Article Title: Targeting mutant NPM1-expressing AML with T cell receptor-like antibodies

doi: 10.1016/j.isci.2026.117630

Figure Lengend Snippet: Engineering and functional characterization of human-mouse chimeric antibodies (A) Schematic diagram of human-mouse chimeric antibodies. (B) SPR analysis revealed a KD of 492 nM for hu2E2 binding to pNPM1-HLA-A2 (six concentrations were injected once each [ N = 1 per concentration] in a single experiment). (C) Detection of the ADCC effect in Jurkat-Lucia NFAT-CD16 cells (response values were plotted against log10(antibody concentration) and fitted by 4 PL nonlinear regression to illustrate the dose-response relationship. N = 3 independent experiments; data are mean ± SD). (D) The anti-tumor activity of the antibodies in vitro was detected via lymphocyte-tumor coculture. ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; N = 5 independent experiments; data are mean ± SD; ordinary one-way ANOVA. (E) ELISA was used to detect the cytokine content. N = 5 independent experiments; data are represented as mean ± SD.

Article Snippet: Human perforin 1 (PRF1) ELISA kit , Elabscience , Cat# E-EL-H1123.

Techniques: Functional Assay, Analysis, Binding Assay, Injection, Concentration Assay, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Targeting mutant NPM1-expressing AML with T cell receptor-like antibodies

doi: 10.1016/j.isci.2026.117630

Figure Lengend Snippet: Diverse formats of engineered antibodies (A) Schematic diagram of 2E2-engineered antibodies. (B) Affinity of hu2E2-CAM16vhh to NK cells, as determined via flow cytometry; affinity of hu2E2-H11vhh for T cells, as determined via flow cytometry (one independent experiment; N = 1). (C) Detection of the ADCC effect in Jurkat-Lucia NFAT-CD16 cells ( N = 3 independent experiments; data are mean ± SD). (D) The anti-tumor activity of the antibodies in vitro was detected via lymphocyte-tumor coculture. ∗ p < 0.05; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001; N = 5 independent experiments; data are mean ± SD; ordinary one-way ANOVA. (E) ELISA was used to detect the cytokine content. N = 5 independent experiments; data are represented as mean ± SD.

Article Snippet: Human perforin 1 (PRF1) ELISA kit , Elabscience , Cat# E-EL-H1123.

Techniques: Flow Cytometry, Activity Assay, In Vitro, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Targeting mutant NPM1-expressing AML with T cell receptor-like antibodies

doi: 10.1016/j.isci.2026.117630

Figure Lengend Snippet: Effect of Fc region mutation of the 2E2 antibody on the killing of AML cells in vitro (A) Model diagram of the introduction of mutations in the Fc region of the 2E2 antibody. (B) Three Fc-Silence antibodies expressed and purified via the CHO expression system were identified via SDS-PAGE (one independent experiment; N = 1). (C) Detection of the ADCC effect in Jurkat-Lucia NFAT-CD16 cells ( N = 3 independent experiments; data are mean ± SD). (D) The antitumor activity of the antibody in vitro was detected via lymphocyte-tumor coculture. ( N = 5 independent experiments for the hu2E2-CAM16vhh-DANA, hu2E2-H11vhh-DANA, and hu2E2-L1H1-DANA groups, and N = 4 for the Control group. Data are shown as mean ± SD). (E) ELISA was used to measure the levels of cytokines. ∗ p < 0.05;∗∗∗∗ p < 0.0001; N = 5 independent experiments; data are mean ± SD; ordinary one-way ANOVA).

Article Snippet: Human perforin 1 (PRF1) ELISA kit , Elabscience , Cat# E-EL-H1123.

Techniques: Mutagenesis, In Vitro, Purification, Expressing, SDS Page, Activity Assay, Control, Enzyme-linked Immunosorbent Assay

Journal: iScience

Article Title: Targeting mutant NPM1-expressing AML with T cell receptor-like antibodies

doi: 10.1016/j.isci.2026.117630

Figure Lengend Snippet:

Article Snippet: Human perforin 1 (PRF1) ELISA kit , Elabscience , Cat# E-EL-H1123.

Techniques: Cell-Signaling, Purification, Recombinant, Sequencing, Control, Plasmid Preparation, Extraction, Transfection, Bicinchoninic Acid Protein Assay, Conjugation Assay, Staining, Enzyme-linked Immunosorbent Assay, Software

Journal: iScience

Article Title: M2 type tumor-associated macrophage-derived CCL-18 promotes glioma cell proliferation but does not mediates glioma recurrence after radiotherapy

doi: 10.1016/j.isci.2026.117695

Figure Lengend Snippet: CCL-18 may be the candidate-mediator of glioma recurrence (A) KEGG enrichment analysis of the 201 DEGs showing that the cytokine-cytokine receptor interaction pathway was the most significantly enriched. (B) Among cytokine-cytokine receptor interaction pathway, CCL-18 was the top one up-regulated DEG. Data were presented as average TPM. (C) Representative IHC staining of CCL-18 in primary and recurrent glioma samples from irradiated patients versus unirradiated patients. Scale bars: 200 μm and 100 μm (enlarged graph). (D and E) Quantification of IHC results in (C). The percent of CCL-18 positive cells in primary and recurrent glioma samples accepting radiotherapy (D), and without radiotherapy (E). Data were presented as paired individual value (two-tailed paired t test, ∗ p < 0.05). (F) The correlation between tumor volume doubling time and the level of CCL-18. Data were presented as Pearson correlation coefficient. (G) Kaplan-Meier curves showing the progression free survival probability of glioma patients accepting radiotherapy based on different expression levels of CCL-18. Data were presented as median ± 95% confidence intervals (∗∗ p < 0.01). Data were obtained from 15 paired primary glioma and recurrent glioma with radiotherapy, and 8 paired primary glioma and recurrent glioma without radiotherapy (C–G).

Article Snippet: CCL-18 ELISA kit , Boster Biological Technology , EK0686.

Techniques: Analysis, Immunohistochemistry, Irradiation, Two Tailed Test, Expressing

Journal: iScience

Article Title: M2 type tumor-associated macrophage-derived CCL-18 promotes glioma cell proliferation but does not mediates glioma recurrence after radiotherapy

doi: 10.1016/j.isci.2026.117695

Figure Lengend Snippet: Multivariate Cox regression analysis from TCGA

Article Snippet: CCL-18 ELISA kit , Boster Biological Technology , EK0686.

Techniques: Analysis, Expressing

Journal: iScience

Article Title: M2 type tumor-associated macrophage-derived CCL-18 promotes glioma cell proliferation but does not mediates glioma recurrence after radiotherapy

doi: 10.1016/j.isci.2026.117695

Figure Lengend Snippet: Radiation promotes macrophage to polarize into M2-TAMs, accompanying CCL-18 secretion (A) The mRNA level of CD163 and IL-10 after treatment with PMA or PMA+IL-4/13 on THP-1 cells. Data were presented as mean ± SEM (unpaired t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (B) Representative immunoblot of CD163 and CD68 after PMA or PMA+IL-4/13 treatment in THP-1 cells. (C) Representative immunoblot of CD163 and CD68 after PMA treatment with or without different radiation doses. (D) The mRNA level of CD163 and IL-10 following the indicated treatment at different durations. Data were presented as mean ± SEM (one-way ANOVA, in CD163: F = 1.22, p = 0.32, ∗∗∗ p < 0.001; in IL-10: F = 1.82, p = 0.14, ∗∗∗ p < 0.001). (E) Representative immunoblot of CD163 and CD68 following the indicated treatment. (F) Representative immunofluorescence images of CD163 and CD68 staining following the indicated treatment. Scale bars: 200 μm. (G and H) Quantitative results of relative mean density of CD163 (G) and CD68 (H). Data were presented as mean ± SEM (one-way ANOVA, in CD163: F = 1.36, p = 0.3, ∗∗ p < 0.01, ∗∗∗ p < 0.001; in CD68: F = 1.54, p = 0.26, ∗ p < 0.05, ∗∗ p < 0.01). (I) Representative immunoblot of indicated antibodies on THP-1 cells after PMA treatment with or without radiation. (J and K) The mRNA level (J) and the protein abundance (K) of CCL-18 following the indicated treatment. Data were presented as mean ± SEM (one-way ANOVA, in mRNA: F = 1.35, p = 0.27, ∗∗ p < 0.01, ∗∗∗ p < 0.001; in protein: F = 3, p = 0.04, ∗∗∗ p < 0.001). Data were obtained from 4 independent experiments (A, F–H, and K), 3 independent experiments (B, C, E, and I) and 6 independent experiments (D and J).

Article Snippet: CCL-18 ELISA kit , Boster Biological Technology , EK0686.

Techniques: Western Blot, Immunofluorescence, Staining, Quantitative Proteomics

Journal: iScience

Article Title: M2 type tumor-associated macrophage-derived CCL-18 promotes glioma cell proliferation but does not mediates glioma recurrence after radiotherapy

doi: 10.1016/j.isci.2026.117695

Figure Lengend Snippet: CCL-18 does not mediate glioma cell proliferation following radiotherapy in vitro (A) Viability of GBM cells after treatment with different concentrations of CCL-18. Data were presented as mean ± SEM (one-way ANOVA, F = 1.9, p = 0.21, ∗∗ p < 0.01, ∗∗∗ p < 0.001). (B) Representative images of colony formation assay after CCL-18 (100 ng/mL) treatment. (C) Quantitative results from (B). Data were presented as mean ± SD (unpaired t test, ∗∗∗ p < 0.001). (D) Schematic workflow for the co-culture experiment. (E) Representative images of colony formation assay. (F) Quantitative results from (E). Data were presented as mean ± SD (unpaired t test, ∗ p < 0.05). (G) The mRNA level of PITPNM3, GPR30, and CCR8 on irradiated GBM cells. Data were presented as mean ± SEM (unpaired t test, ∗∗∗ p < 0.001). (H) Representative immunoblot of PITPNM3, GPR30, and CCR8 on irradiated GBM cells. (I) Quantitative results from (H). Data were presented as mean ± SEM (unpaired t test, ∗ p < 0.05, ∗∗ p < 0.01). (J) Immunoblot of CCR8 after overexpression. (K) Representative images of colony formation assay on irradiated GBM cells after CCL-18 (100 ng/mL) treatment. (L) Quantitative results from (K). Data were presented as mean ± SD (two-way ANOVA, F = 4.9, p = 0.06, ∗ p < 0.05, ∗∗ p < 0.01). Data were obtained from 3 independent experiments (A–L).

Article Snippet: CCL-18 ELISA kit , Boster Biological Technology , EK0686.

Techniques: In Vitro, Colony Assay, Co-culture Assay, Irradiation, Western Blot, Over Expression

Journal: iScience

Article Title: M2 type tumor-associated macrophage-derived CCL-18 promotes glioma cell proliferation but does not mediates glioma recurrence after radiotherapy

doi: 10.1016/j.isci.2026.117695

Figure Lengend Snippet: CCL-18 do not mediate glioma recurrence following radiotherapy in vivo (A) Viability of GL261 cells after treatment with different concentrations of CCL-18. Data were presented as mean ± SEM (one-way ANOVA, F = 0.9, p = 0.48, ∗∗∗ p < 0.001). (B) Viability of GL261 cells after treatment with CCL-18 (100 ng/mL) and R243 (50, 100 μM). Data were presented as mean ± SEM (one-way ANOVA, F = 1.43, p = 0.28, ∗ p < 0.05, ∗∗∗ p < 0.001). (C) Representative immunoblot of ACP5, Akt, and phosphorylated Akt on GL261 cells after CCL-18 (100 ng/mL) treatment. (D) Schematic workflow for the cell-derived allograft experiment. (E) The bioluminescence value in mice receiving radiation on the indicated days. Data were presented as mean ± SEM (one-way ANOVA, in radiation: F = 2.48, p = 0.1, ∗ p < 0.05; in radiation with CCL-18 treatment: F = 1.41, p = 0.3, ∗ p < 0.05). (F) Representative pseudo-color bioluminescence images of intracranial allografts receiving radiation with or without CCL-18 administration. (G) Quantitative results from (F) on the indicated days. Data were presented as mean ± SEM (two-way ANOVA, F = 2.39, p = 0.04, ∗ p < 0.05, ∗∗ p < 0.01). (H) Kaplan-Meier curve showing the percent survival of mice receiving different treatment. Data were presented as median (∗∗∗ p < 0.001). (I) The percent of M1-TAM in glioma after radiation with or without CCL-18 treatment. (J) Quantitative results from (I). Data were presented as mean ± SD (one-way ANOVA, F = 1.46, p = 0.26). (K)The percent of M2-TAM in glioma after radiation with or without CCL-18 treatment. (L) Quantitative results from (K). Data were presented as mean ± SD (one-way ANOVA, F = 0.1, p = 0.9). Data were obtained from 3 independent experiments (A, C, and I–L), and 4 independent experiments (B). Data were obtained from ≥6 mice (E), ≥11 mice (G and H), and 3 mice (I–L).

Article Snippet: CCL-18 ELISA kit , Boster Biological Technology , EK0686.

Techniques: In Vivo, Western Blot, Derivative Assay

Journal: iScience

Article Title: M2 type tumor-associated macrophage-derived CCL-18 promotes glioma cell proliferation but does not mediates glioma recurrence after radiotherapy

doi: 10.1016/j.isci.2026.117695

Figure Lengend Snippet:

Article Snippet: CCL-18 ELISA kit , Boster Biological Technology , EK0686.

Techniques: Cell-Signaling, Virus, Tissue, Microarray, Recombinant, cDNA Synthesis, Enzyme-linked Immunosorbent Assay, Software, Biomarker Discovery, In Vivo Imaging

Journal: MedComm

Article Title: Blocking Extracellular Cyclophilin A‐Mediated Adhesion Protects Against Bacterial Infection

doi: 10.1002/mco2.71063

Figure Lengend Snippet: eCypA is critical for the adhesion of GAS and S.P (A) C57BL/6 mice ( n = 7) were infected intranasally with PBS or 3000 PFU of influenza A/WSN/33 (H1N1) on Day 0 followed by intraperitoneal injection of isotype IgG (IgG) or anti‐CypA mAb (100 µg/mouse) on Day 1, and then infected intranasally with GAS (1.0 × 10 7 CFU), S. P (1.0 × 10 7 CFU), K. P (1.0 × 10 7 CFU), or PBS on Day 3. (B) Adherence of GAS in the lungs of C57BL/6 mice on Day 4. (C) Adherence of S. P in the lungs of C57BL/6 mice on Day 4. (D) The lung index (100× lung/body weight) was calculated on Day 4. (E) The body weights of C57BL/6 mice were monitored during Days 0–4. (F) Hematoxylin and eosin staining of mouse lungs on Day 4 (left). The severity of lung injury was analyzed in a blinded manner (right). Scale bar, 150 µm. (G) Heatmap showing the results of the qPCR analysis indicating the mRNA levels of proinflammatory cytokines in the lungs of C57BL/6 mice on Day 4. (H and I) Adherence of GAS (H) and S. P (I) in A549 cells infected with PBS or IAV (MOI = 1) followed by treatment with IgG or anti‐CypA mAb (50 nM) for 24 h and then infection of the cells with GAS (MOI = 10) for 2 h. Data are reported as the percentage of adherent bacteria relative to the input inoculum. (J) C57BL/6 mice were preinjected intraperitoneally with eCypA (40 µg/mouse) or vehicle (PBS) for 2 h, followed by intraperitoneal injection with anti‐CypA mAb (100 µg/mouse) or vehicle (PBS) on Day 0 and then infected intranasally with GAS (1.0 × 10 7 CFU), S. P (1.0 × 10 7 CFU), K. P (1.0 × 10 7 CFU), or PBS on Day 3. (K) ELISA of eCypA in BALF from mice infected with GAS. (L–N) Adherence of GAS (K), S. P (L), and K. P (M) in the lungs of C57BL/6 mice ( n = 5) on Day 4. (O–Q) Adherence assays of GAS (N), S. P (O), and K. P (P) in A549 cells pretreated with PBS, eCypA (10 nM) or eCypA + anti‐CypA mAb (10 nM eCypA + 50 nM anti‐CypA mAb, preincubated at 37°C for 1 h) for 12 h, followed by GAS, S. P , or K. P (MOI = 10) infection for 2 h. Data are shown as the percentage of adherent bacteria relative to the input inoculum. Data shown in (B–F) and (K–N) are presented as the means ± SEMs; Data shown in (H), (I), and (O–Q) are presented as the means ± SDs; * p < 0.05, ** p < 0.01, *** p < 0.001 and ns = not significant. Unpaired two‐tailed Student's t test for (B) and (C), and one‐way ANOVA followed by Dunnett's test for (D–F), (H), (I), and (K–Q).

Article Snippet: eCypA concentrations in BALF, serum, and cell culture supernatants were quantified by ELISA (CSB‐E09920h, CSB‐E09282m, CUSABIO ).

Techniques: Infection, Injection, H&E Stain, Analysis, Bacteria, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: MedComm

Article Title: Blocking Extracellular Cyclophilin A‐Mediated Adhesion Protects Against Bacterial Infection

doi: 10.1002/mco2.71063

Figure Lengend Snippet: Binding of eCypA to TβR2 facilitates the formation of the TGFβ1‐TGF‐β receptor 2 complex (A) qPCR analysis of ITGA5 and FN mRNA levels in A549 cells transfected with siRNAs targeting TβR2, CD147, ACE2, or MUC18 for 48 h, and then treated with 10 nM eCypA or PBS for 12 h. (B) Immunoblot analysis of the indicated proteins in immunoprecipitated samples and whole‐cell lysates (WCL) of A549 cells treated with Fc, CypA‐Fc (200 ng/mL), R55‐Fc (200 ng/mL), CypA‐Fc + anti‐CypA mAb (1 µg/mL), or R55‐Fc + anti‐CypA mAb for 2 h. (C) Interactions of CypA with TGF‐β1, TβR1, and TβR2 were detected using ELISA. BSA was used as a negative control. (D) The blocking effect of the anti‐CypA mAb on the binding of CypA to TβR2 was detected using ELISA. (E–H) Interaction of CypA with anti‐CypA mAb (E) and TβR2 (F) was detected by SPR. The competitive effect of anti‐CypA mAb on CypA‐TβR2 interaction was detected using competitive SPR assay (G). Linearities of the CypA‐TβR2 interaction were generated from the data of the sampling points shown in (H). (I) Immunoblot analysis of the indicated proteins in A549 cells treated with eCypA (10 nM) or eCypA (10 nM) + TGF‐β pAb (50 nM) for 30 min. (J) Adherence assays of GAS in A549 cells treated with eCypA (10 nM) or eCypA (10 nM) + TGF‐β pAb (50 nM) for 12 h, and then infected with GAS (MOI = 10.0) for 2 h. Data are reported as the percentage of adherent bacteria relative to the input inoculum. (K) Immunoblot analysis of the indicated proteins in immunoprecipitated samples and WCLs of A549 cells treated with TGF‐β1 (5 ng/mL), TGF‐β1 + CypA‐His (10 nM), or TGF‐β1 + CypA‐His + anti‐CypA mAb (50 nM) for 2 h. (L and M) Interactions of TGF‐β1 with TβR2 were detected by SPR in the absence (L) or presence (M) of CypA. Data are presented as the means ± SDs; ** p < 0.01, *** p < 0.001 and ns = not significant; unpaired two‐tailed Student's t test for (D); one‐way ANOVA followed by Dunnett's test for (A) and (J).

Article Snippet: eCypA concentrations in BALF, serum, and cell culture supernatants were quantified by ELISA (CSB‐E09920h, CSB‐E09282m, CUSABIO ).

Techniques: Binding Assay, Analysis, Transfection, Western Blot, Immunoprecipitation, Enzyme-linked Immunosorbent Assay, Negative Control, Blocking Assay, SPR Assay, Generated, Sampling, Infection, Bacteria, Two Tailed Test

Journal: Advanced Science

Article Title: Lactylation‐Mediated HER2 Stabilization Promotes Trastuzumab Resistance in Gastric Cancer

doi: 10.1002/advs.77955

Figure Lengend Snippet: Lactate promotes trastuzumab resistance in gastric cancer by stabilizing HER2 expression. (a) Volcano plot illustrating metabolic differences between gastric cancer tissues from patients who subsequently developed trastuzumab resistance ( n = 6) and those who remained trastuzumab‐sensitive ( n = 6), as determined by untargeted metabolomics analysis. All tissues were surgically resected prior to trastuzumab treatment, and sensitivity or resistance to trastuzumab was subsequently determined based on clinical pathology data and patient follow‐up information. Red dots represent metabolites involved in the glycolytic pathway. (b) Lactate levels in gastric cancer specimens measured using a colorimetric assay kit (all samples assayed in duplicate). Each patient sample is shown in a distinct color, and the two technical replicates from the same patient are shown in the same color. (c) HER2 protein levels measured by enzyme‐linked immunosorbent assay (ELISA) in gastric cancer samples (LAC low , n = 6; LAC High , n = 6). (d,e) HER2 protein levels in AGS cells following treatment with sodium lactate (NALA, 25 or 50 m m ) and lactate (10 or 20 m m ). (f,g) HER2 protein levels in AGS cells following treatment with sodium oxamate (10 or 20 m m ) and stiripentol (100 or 200 µ m ). (h,i) HER2 protein levels in NCI‐N87 cells following treatment with NALA (25 or 50 m m ) and lactate (10 or 20 m m ). (j,k) HER2 protein levels in NCI‐N87 cells following treatment with sodium oxamate (10 or 20 m m ) and stiripentol (100 or 200 µ m ). (l) Flow cytometric analysis of HER2 protein expression in AGS cells treated with NALA or lactate dehydrogenase inhibitors (LDHi) ( n = 3). (m) Flow cytometric analysis of HER2 protein expression in NCI‐N87 cells treated with NALA or LDHi ( n = 3). (n) Representative immunofluorescence (IF) staining and co‐localization analysis of pan‐lactylation and HER2 in gastric cancer tissues (scale bars, 20 µm). (o) Correlation between pan‐lactylation and HER2 expression in gastric cancer tissues (Spearman correlation analysis). (p,q) Lactylation levels of exogenous and endogenous HER2 in cells treated with NALA or LDHi. (r,s) HER2 protein levels in 293T and NCI‐N87 cells treated with LDHi and/or MG132 (10 µ m ). (t,u) Ubiquitination of exogenous and endogenous HER2 following NALA treatment. (v,w) Ubiquitination of exogenous and endogenous HER2 following LDHi treatment. p ‐values were determined by Student's t ‐test; ns, not significant. Data are presented as mean ± SD.

Article Snippet: The concentration of HER2 was measured using a commercial ELISA kit (Elabscience E‐EL‐H6083) according to the product's instructions.

Techniques: Expressing, Analysis, Sensitivity, Pathology, Colorimetric Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Ubiquitin Proteomics

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: Additionally, the commercial ELISA kits (Human and mouse MPO ELISA Kits, Cat. # CSB-E08721h, CSB-E08723m, CUSABIO, Wuhan, China; Human and mouse neutrophil elastase ELISA Kit, Cat. # CSB-EL007587HU, CSB-E04804m, CUSABIO) were applied to quantify the levels of MPO and neutrophil elastase released into cell culture supernatants as per the instructions of the manufacturer.

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 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: Additionally, the commercial ELISA kits (Human and mouse MPO ELISA Kits, Cat. # CSB-E08721h, CSB-E08723m, CUSABIO, Wuhan, China; Human and mouse neutrophil elastase ELISA Kit, Cat. # CSB-EL007587HU, CSB-E04804m, CUSABIO) were applied to quantify the levels of MPO and neutrophil elastase released into cell culture supernatants as per the instructions of the manufacturer.

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: Additionally, the commercial ELISA kits (Human and mouse MPO ELISA Kits, Cat. # CSB-E08721h, CSB-E08723m, CUSABIO, Wuhan, China; Human and mouse neutrophil elastase ELISA Kit, Cat. # CSB-EL007587HU, CSB-E04804m, CUSABIO) were applied to quantify the levels of MPO and neutrophil elastase released into cell culture supernatants as per the instructions of the manufacturer.

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: Additionally, the commercial ELISA kits (Human and mouse MPO ELISA Kits, Cat. # CSB-E08721h, CSB-E08723m, CUSABIO, Wuhan, China; Human and mouse neutrophil elastase ELISA Kit, Cat. # CSB-EL007587HU, CSB-E04804m, CUSABIO) were applied to quantify the levels of MPO and neutrophil elastase released into cell culture supernatants as per the instructions of the manufacturer.

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 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: Additionally, the commercial ELISA kits (Human and mouse MPO ELISA Kits, Cat. # CSB-E08721h, CSB-E08723m, CUSABIO, Wuhan, China; Human and mouse neutrophil elastase ELISA Kit, Cat. # CSB-EL007587HU, CSB-E04804m, CUSABIO) were applied to quantify the levels of MPO and neutrophil elastase released into cell culture supernatants as per the instructions of the manufacturer.

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: ZDHHC5 overexpression partially counteracts the protective effects of NOD2 knockdown in a mouse model of COPD The ability of ZDHHC5 overexpression to counteract the effects of NOD2 knockdown was evaluated in a cigarette smoke (CS)-induced mouse model of COPD. ( A ) Schematic of the in vivo experimental design. ( B ) qRT-PCR analysis of NOD2 and ZDHHC5 mRNA expression in lung tissue. ( C ) Representative images of H&E staining of lung sections. Scale bar: 100 μm. ( D ) Representative images of Masson’s trichrome staining for collagen deposition. Scale bar: 100 μm. ( E ) Quantification of inflammatory cells and TNF-α, IL-6, and CXCL1 levels in BALF. ( F ) Representative images of IHC staining for E-cadherin and N-cadherin. Scale bar: 50 μm. ( G ) Quantitative analysis of IHC staining. ( H ) Immunofluorescence images showing spatial overlap between Ly6G (green) and LC3B (red). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. ( I ) Immunofluorescence images showing NETs stained for CitH3 (green) and MPO (red). Scale bars: 20 μm in the upper panels and 10 μm in the enlarged lower panels. ( J ) Quantification of CitH3-DNA and MPO-DNA complexes in serum and lung tissue homogenates by ELISA. Data are presented as mean ± SD. Quantitative data were analyzed using two-way ANOVA followed by Tukey’s test. * p < 0.05, ** p < 0.01 vs. COPD + sh-NC + Vector group; # p < 0.05, ## p < 0.01 vs. COPD + sh-NOD2 + ZDHHC5 group

Article Snippet: Additionally, the commercial ELISA kits (Human and mouse MPO ELISA Kits, Cat. # CSB-E08721h, CSB-E08723m, CUSABIO, Wuhan, China; Human and mouse neutrophil elastase ELISA Kit, Cat. # CSB-EL007587HU, CSB-E04804m, CUSABIO) were applied to quantify the levels of MPO and neutrophil elastase released into cell culture supernatants as per the instructions of the manufacturer.

Techniques: Over Expression, Knockdown, In Vivo, Quantitative RT-PCR, Analysis, Expressing, Tissue, Staining, Immunohistochemistry, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Plasmid Preparation