Journal: Autophagy Reports

Article Title: LC3-associated phagocytosis is impaired in monocyte-derived macrophages from systemic sclerosis patients

doi: 10.1080/27694127.2026.2730912

Figure Lengend Snippet: Reagent list and references.

Article Snippet: Pan T cell Isolation Kit, human , – , Miltenyi Biotec, 130-096-535.

Techniques: Concentration Assay, Cell Isolation, Luminex, Glass, Flow Cytometry

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:

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: Advanced Science

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

doi: 10.1002/advs.77955

Figure Lengend Snippet: AARS1‐mediated HER2 K1171 lactylation enhances its protein stability. (a) Identification of candidate proteins for HER2 lactylation by liquid chromatography‐mass spectrometry (LC‐MS/MS). (b) Screening for the “writer” of HER2 lactylation using co‐immunoprecipitation (Co‐IP). (c) Exogenous Co‐IP assay confirming the interaction between HER2 and AARS1 in 293T cells. (d) Endogenous Co‐IP assays confirming the interaction in NCI‐N87 cells. (e) In vitro lactylation assay showing that AARS1 directly mediates HER2 lactylation. (f,g) IF staining showing the co‐localization of HER2 and AARS1 in human gastric cancer tissue (scale bars, 50 µm) (f) and NCI‐N87 cells (scale bars, 5 µm) (g). (h) Integrating the lactylation sites identified by LC‐MS/MS with DeepKla predictions. (i) Identification of the lactylation site in HER2. (j) Conservation analysis of the lactylation site in HER2 orthologs across species. (k) Western blot analysis of HER2 lactylation in NCI‐N87 cells treated with NALA or LDHi. (l–n) HER2 K1171 lactylation levels following AARS1 overexpression (l), AARS1 knockdown (m), and L‐alanine treatment (n). (o) Lactylation levels of HER2 wild‐type (WT) and the K1171R mutant following AARS1 overexpression. (p,q) Protein levels of HER2 WT and HER2 K1171R mutant following NALA or LDHi treatment. (r–t) Protein levels of HER2 WT and HER2 K1171R mutant following AARS1 overexpression (r), AARS1 knockdown (s), and L‐alanine treatment (t). (u) Western blot analysis of ubiquitination levels in HER2 and the K1171 mutant. (v–x) Ubiquitination of HER2 WT and HER2 K1171R mutant following AARS1 overexpression (v), AARS1 knockdown (w), and L‐alanine treatment (x).

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

Techniques: Stability, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Screening, Co-Immunoprecipitation Assay, In Vitro, Staining, Tissue, Analysis, Western Blot, Over Expression, Knockdown, Mutagenesis, Ubiquitin Proteomics

Journal: Advanced Science

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

doi: 10.1002/advs.77955

Figure Lengend Snippet: K1171 lactylation of HER2 promotes its interaction with USP11. (a,b) HER2 protein levels in 293T and NCI‐N87 cells treated with NALA and/or PR619 (10 µ m ). (c) Deubiquitinases (DUBs) interacting with HER2 identified by LC‐MS/MS analysis and ranked by enrichment score. (d) Knockout of the nine candidate DUBs to identify the key regulator of HER2 protein stability. (e) Western blot analysis of HER2 protein levels in NCI‐N87 cells following USP11 knockout. (f) Western blot analysis of HER2 protein levels in NCI‐N87 cells following USP11 overexpression. (g) HER2 protein levels following overexpression of USP11 WT and its inactive mutant. (h) Ubiquitination levels of HER2 in 293T cells following USP11 overexpression. (i) Ubiquitination levels of HER2 following overexpression of USP11 WT and its inactive mutant. (j,k) Exogenous Co‐IP assays confirming the interaction between HER2 and USP11 in 293T cells. (l) Endogenous Co‐IP assay confirming the interaction between HER2 and USP11 in NCI‐N87 cells. (m) IF staining showing the co‐localization of HER2 and USP11 in NCI‐N87 cells (scale bars, 5 µm). (n,o) Co‐IP analysis of the interaction between MYC‐HER2 and HA‐USP11 following NALA (n) and LDHi (o) treatment. (p–r) Co‐IP analysis of the interaction between HER2 and USP11 binding following AARS1 overexpression (p), AARS1 knockdown (q), and L‐alanine treatment (r). (s) Co‐IP analysis of USP11 binding to HER2 WT and the K1171R mutant. (t) Protein levels of HER2 WT and HER2 K1171R mutant following USP11 overexpression.

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

Techniques: Liquid Chromatography with Mass Spectroscopy, Analysis, Knock-Out, Stability, Western Blot, Over Expression, Mutagenesis, Ubiquitin Proteomics, Co-Immunoprecipitation Assay, Staining, Binding Assay, Knockdown

Journal: Advanced Science

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

doi: 10.1002/advs.77955

Figure Lengend Snippet: AARS1‐mediated HER2 lactylation promotes its interaction with EGFR. (a,b) The interaction between exogenously expressed HER2 and EGFR in 293T cells treated with NALA (a) and LDHi (b). (c–e) Co‐IP analysis of endogenous HER2‐EGFR binding in NCI‐N87 cells following NALA (c), LDHi (d), and L‐alanine treatment (e). (f) In vitro GST pull‐down assay showing the effect of AARS1‐induced HER2 lactylation on HER2‐EGFR binding. Coomassie blue staining of purified AARS1 used in the pull‐down assay. (g) Representative IF images and quantitative analysis of HER2 and EGFR in NCI‐N87 cells ( n = 3) following NALA and LDHi treatment (scale bars, 5 µm). (h) Co‐IP analysis of EGFR binding to HER2 WT and HER2 K1171 mutant following NALA treatment. (i) Co‐IP analysis of EGFR binding to HER2 WT and HER2 K1171 mutant following AARS1 overexpression. (j) Representative IF images and quantitative analysis of HER2 K1171 lactylation and EGFR in NCI‐N87 cells ( n = 3) following NALA and LDHi treatment (scale bars, 5 µm). 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: Co-Immunoprecipitation Assay, Analysis, Binding Assay, In Vitro, GST Pulldown Assay, Staining, Purification, Pull Down Assay, Mutagenesis, Over Expression

Journal: Advanced Science

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

doi: 10.1002/advs.77955

Figure Lengend Snippet: Lactate suppression improves trastuzumab efficacy in gastric cancer. (a) Schematic of luciferase‐labeled NCI‐N87 xenograft mouse model establishment. (b) Representative in vivo bioluminescence images of luciferase‐labeled gastric cancer xenografts ( n = 5 per group). All images were acquired under identical exposure parameters and displayed on the same color scale (10,000–60,000). (c) Quantification of tumor burden by region of interest (ROI) analysis over the observation period. (d) Bioluminescence intensity of individual tumors at the end of treatment ( n = 5). (e,f) Flow cytometric analysis and quantification of HER2 expression in mouse tumor tissues ( n = 5). (g,h) Flow cytometric analysis and quantification of EdU incorporation in mouse tumor tissues ( n = 5). (i,j) Representative immunohistochemical (IHC) staining images (scale bars, 50 µm) and quantitative analysis of HER2‐K1171la in mouse tumor tissues ( n = 5, four random fields per tissue section were analyzed). (k,l) Tumor growth curves and Kaplan‐Meier survival analysis of all treatment groups in the NCI‐N87 gastric cancer xenograft model ( n = 6; log‐rank test). (m) Representative images of dissected tumors from the HER2‐overexpressing AGS xenograft model. (n) Tumor volume monitoring in different treatment groups. (o) Volume measurement of dissected tumor ( n = 6). (p) Weight measurement of dissected tumor ( n = 6). p ‐values were determined by two‐way ANOVA (c and n), mixed‐effects models (k), Student's t ‐test (d, f, h, o, and p), or Mann‐Whitney U test (j); 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: Luciferase, Labeling, Xenograft Assay, In Vivo, Image Analysis and Quantification, Analysis, Expressing, EdU Assay, Immunohistochemical staining, Immunohistochemistry, Tissue, MANN-WHITNEY

Journal: Advanced Science

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

doi: 10.1002/advs.77955

Figure Lengend Snippet: Inhibition of HER2 lactylation overcomes trastuzumab resistance in gastric cancer. (a) Representative images of dissected tumors from the NCI‐N87 xenograft model. (b) Tumor volume monitoring in different treatment groups. (c) End‐point volume of dissected tumors ( n = 6). (d) End‐point weight of dissected tumors ( n = 6). (e) Representative images of dissected tumors from the XELOX‐based combination therapy. (f) Tumor volume monitoring in different treatment groups. (g) End‐point volume of dissected tumors ( n = 6). (h) End‐point weight of dissected tumors ( n = 6). (i,j) Representative IHC staining images (scale bars, 50 µm) and quantification of Ki‐67 positive area in mouse tumor tissues ( n = 6, four random fields per tissue section were analyzed). (k) Schematic illustration of the trastuzumab‐resistant gastric cancer mouse model establishment. (l) Representative images of dissected tumors from the resistant xenograft model. (m) Tumor volume curves in the trastuzumab‐resistant model. (n) End‐point volume in the resistant model ( n = 6). (o) End‐point weight in the resistant model ( n = 6). (p,q) Flow cytometric analysis of HER2 expression in tumors from the resistant model ( n = 6). (r,s) Flow cytometric measurement of EdU incorporation in tumors from the resistant model ( n = 6). (t,u) HER2‐K1171la IHC staining (scale bars, 50 µm) and quantification in the resistant model ( n = 6, four random fields per tissue section were analyzed). XELOX, capecitabine plus oxaliplatin. p ‐values were determined by two‐way ANOVA (b, f, and m), Student's t ‐test (d, g, h, and n), Welch's‐ t test (s) or Mann‐Whitney U test (j, o, q, and u); 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: Inhibition, Xenograft Assay, Immunohistochemistry, Tissue, Analysis, Expressing, EdU Assay, MANN-WHITNEY

Journal: Advanced Science

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

doi: 10.1002/advs.77955

Figure Lengend Snippet: Targeting HER2 K1171 lactylation with a peptide inhibitor enhances trastuzumab sensitivity. (a) Schematic illustration of the designed peptide. Green represents the cell‐penetrating peptide (CPP), and red indicates the site for HER2 lactylation. (b,c) Western blot analysis of HER2 lactylation and protein expression in NCI‐N87 cells treated with the peptides. (d,e) Western blot analysis of HER2 lactylation and protein expression in NCI‐N87 cells treated with the peptide‐1# (K1171‐pe). (f) Co‐IP analysis of the binding between endogenous HER2 and EGFR in NCI‐N87 cells treated with K1171‐pe and control peptide (K1171R‐pe). (g) Experimental schematic of the NCI‐N87 xenograft mouse model. (h) Representative images of dissected tumors from the NCI‐N87 xenograft model. (i) Tumor volume monitoring in different treatment groups. (j) Endpoint volume of dissected tumors ( n = 6). (k) Endpoint weight of dissected tumors ( n = 6). (l) Experimental schematic of the patient‐derived xenograft (PDX) model. (m) Representative images of dissected tumors from the PDX model. (n) Tumor growth curves in the PDX model. (o) End‐point volume in the PDX model ( n = 6). (p) End‐point weight in the PDX model ( n = 6). (q,r) Representative IHC staining images (scale bars, 50 µm) and quantification of Ki‐67 positive area in mouse tumor tissues ( n = 6, four random fields per tissue section were analyzed). (s) Representative images of dissected tumors from the HER2‐low expression model. (t) Tumor growth curves in the HER2‐low expression model. (u) End‐point tumor volume in the HER2‐low expression model ( n = 6). (v) End‐point tumor weight in the HER2‐low expression model ( n = 6). (w,x) Representative IHC staining images (scale bars, 50 µm) and quantification of Ki‐67 positive area in mouse tumor tissues ( n = 6, four random fields per tissue section were analyzed). p ‐values were determined by two‐way ANOVA (I, n, and t), Student's t ‐test (j, k, o, p, u, and v), or Mann‐Whitney U test (h, r, and x); 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: Sensitivity, Western Blot, Analysis, Expressing, Co-Immunoprecipitation Assay, Binding Assay, Control, Xenograft Assay, Derivative Assay, Immunohistochemistry, Tissue, MANN-WHITNEY

Journal: Advanced Science

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

doi: 10.1002/advs.77955

Figure Lengend Snippet: HER2 K1171 lactylation is increased in trastuzumab‐resistant tumors and associated with poor prognosis. (a) Representative IF images and quantitative analysis of HER2 K1171 lactylation in trastuzumab‐sensitive and trastuzumab‐resistant gastric cancer tissues (scale bars, 100 µm; n = 6). (b) Representative IHC staining images of human gastric cancer tissues (scale bars, 50 µm). (c–e) Correlation analyses of HER2 expression with LDHA expression (c), AARS1 expression (d), and USP11 expression (e) in gastric cancer tissues ( n = 31; Spearman's rank correlation analysis). (f–h) Correlation analyses of HER2 lactylation with LDHA expression (f), AARS1 expression (g), and USP11 expression (h) in gastric cancer tissues ( n = 31; Spearman's rank correlation analysis). (i) Multicolor IF staining of HER2, LDHA, AARS1, USP11, and HER2 lactylation in gastric cancer tissue microarrays (scale bars, 200 µm; 50 µm for magnified insets). (j–l) Correlation analyses of HER2 expression with LDHA expression (j), AARS1 expression (k), and USP11 expression (l) in the tissue microarray cohort ( n = 96; Spearman's rank correlation analysis). (m–o) Correlation analyses of HER2 lactylation with LDHA expression (m), AARS1 expression (n), and USP11 expression (o) in the tissue microarray cohort ( n = 96; Spearman's rank correlation analysis). (p) Quantification of LDHA expression in gastric cancer tissues across American Joint Committee on Cancer (AJCC) stages I‐IV ( n = 96). (q) Quantification of HER2 lactylation across AJCC stages I‐IV ( n = 96). (r,s) Kaplan‐Meier survival curves showing that high LDHA expression (r) and high HER2 K1171 lactylation (s) are associated with poor overall survival ( n = 96, 48 patients per group; log‐rank test). High and low expression groups were defined based on the median cutoff values (LDHA score: 37.4612; HER2 K1171 lactylation: 26.9943). No cases were censored. p ‐values were determined by Student's t ‐test (a, d, f, and g) or Mann‐Whitney U test (p and q); 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: Analysis, Immunohistochemistry, Expressing, Staining, Tissue, Microarray, MANN-WHITNEY

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