Journal: Journal of Inflammation Research

Article Title: HMGCS2 Palmitoylation Promotes Diabetes-Induced Erectile Dysfunction Through PPARα-Mediated Macrophage Extracellular Trap Formation

doi: 10.2147/JIR.S623377

Figure Lengend Snippet: Single-cell transcriptomic analysis uncovers macrophages as a key immune cell population in DMED. ( A ) t-SNE visualizing the annotated cell populations across 60,232 cells in corpus cavernosum from 5 patients with DMED and 3 males with normal erections. ( B ) Bubble diagram showing the expression of the used marker genes across the cell populations. ( C ) t-SNE visualizing the cell population distribution in DMED and control groups, respectively. ( D ) The immune activity score of the cell populations based on the immune-related genes. ( E ) Comparison of the immune activity score of the cell populations between DMED and control groups. ( F and G ) Macrophages-mediated communication network in ( F ) DMED and ( G ) control groups. ( H and I ) Ligand-receptor pairs in macrophages-mediated communications in ( H ) DMED and ( I ) control groups. ( J ) Volcano diagram depicting the DEGs in macrophages between DMED and control groups. ( K and L ) Biological processes and KEGG pathways enriched by the DEGs. **, P<0.01; ***, P<0.001 derived from Wilcoxon test.

Article Snippet: Primary antibodies included: HMGCS2 (ab305,227; Abcam, USA), peptidyl arginine deiminase 4 (PAD4; 17,373-1-AP; Proteintech, China), macrophage elastase (ME; 22,989-1-AP; Proteintech), MPO (22,225-1-AP; Proteintech), GAPDH (T0004; Affinity, USA), β-Tubulin (66,240-1-Ig; Proteintech), and PPARα (66,826-1-Ig; Proteintech).

Techniques: Single Cell, Analysis, Immune Cell, Expressing, Marker, Control, Activity Assay, Comparison, Derivative Assay

Journal: Journal of Inflammation Research

Article Title: HMGCS2 Palmitoylation Promotes Diabetes-Induced Erectile Dysfunction Through PPARα-Mediated Macrophage Extracellular Trap Formation

doi: 10.2147/JIR.S623377

Figure Lengend Snippet: HG and PA treatments induce high expression of HMGCS2 and MET formation in macrophages. ( A and B ) Volcano and heatmap diagrams depicting the DEGs in corpus cavernosum between DMED rats and controls. ( C ) Heatmap visualizing the top 20 up- and downregulated DEGs in DMED, respectively. ( D and E ) Biological processes and KEGG pathways enriched by the DEGs. ( F ) Visualization of PPAR signaling pathway enriched by the DEGs. ( G ) qRT-PCR for detecting the mRNA expression of HMGCS2 in control RAW264.7 macrophages and those with treatments of 25 mmol/L HG and 60 μM PA for 48 h. ( H ) Representative Western blot of HMGCS2, PAD4, ME, and MPO in control and HG- and PA-induced RAW264.7 macrophages. ( I-L ) Quantification of the expression levels of HMGCS2, PAD4, ME, and MPO in control and HG- and PA-induced RAW264.7 macrophages. ***, P<0.001; ****, P<0.0001 derived from Student’s t test.

Article Snippet: Primary antibodies included: HMGCS2 (ab305,227; Abcam, USA), peptidyl arginine deiminase 4 (PAD4; 17,373-1-AP; Proteintech, China), macrophage elastase (ME; 22,989-1-AP; Proteintech), MPO (22,225-1-AP; Proteintech), GAPDH (T0004; Affinity, USA), β-Tubulin (66,240-1-Ig; Proteintech), and PPARα (66,826-1-Ig; Proteintech).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Derivative Assay

Journal: Journal of Inflammation Research

Article Title: HMGCS2 Palmitoylation Promotes Diabetes-Induced Erectile Dysfunction Through PPARα-Mediated Macrophage Extracellular Trap Formation

doi: 10.2147/JIR.S623377

Figure Lengend Snippet: HG and PA treatments induce ROS over-production, oxidative stress, and mitochondrial dysfunction in macrophages. ( A ) Representative intracellular ROS photographs using DCFH-DA fluorescence probe in control RAW264.7 macrophages and those with treatments of 25 mmol/L HG and 60 μM PA for 48 h. Scale bars, 50 μm. ( B ) Quantification of fluorescence intensity of ROS in control and HG- and PA-induced RAW264.7 macrophages. ( C and D ) Measurement of MDA and GSH levels in control and HG- and PA-induced RAW264.7 macrophages. ( E ) Representative JC-1 staining photographs in control and HG- and PA-induced RAW264.7 macrophages. Green fluorescence represents that MMP is low and JC-1 exists in the cytoplasm in the form of monomer. Red fluorescence represents that MMP is high and JC-1 polymer is formed. Scale bars, 50 μm. ( F ) Calculation of a ratio of green fluorescence and red fluorescence for quantification MMP changes. ( G ) Detection of ATP in control and HG- and PA-induced RAW264.7 macrophages. ( H ) TEM for investigating the changes in mitochondrial morphology in control and HG- and PA-induced RAW264.7 macrophages. Scale bars, 500 nm. **, P<0.01; ***, P<0.001; ****, P<0.0001 derived from Student’s t test.

Article Snippet: Primary antibodies included: HMGCS2 (ab305,227; Abcam, USA), peptidyl arginine deiminase 4 (PAD4; 17,373-1-AP; Proteintech, China), macrophage elastase (ME; 22,989-1-AP; Proteintech), MPO (22,225-1-AP; Proteintech), GAPDH (T0004; Affinity, USA), β-Tubulin (66,240-1-Ig; Proteintech), and PPARα (66,826-1-Ig; Proteintech).

Techniques: Fluorescence, Control, Staining, Polymer, Morphology, Derivative Assay

Journal: Journal of Inflammation Research

Article Title: HMGCS2 Palmitoylation Promotes Diabetes-Induced Erectile Dysfunction Through PPARα-Mediated Macrophage Extracellular Trap Formation

doi: 10.2147/JIR.S623377

Figure Lengend Snippet: Pharmacological suppression of protein palmitoylation downregulates HMGCS2 palmitoylation and expression and hinders MET formation in HG- and PA-induced macrophages. ( A ) ABE assay for measuring HMGCS2 palmitoylation in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment, in the presence or absence of HAM. ( B ) Co-IP assay for detecting the interaction of HMGCS2 with PPARα in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. ( C ) qRT-PCR for measuring the mRNA level of HMGCS2 in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. ( D ) Representative Western blot of HMGCS2, PAD4, ME, and MPO in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. ( E–H ) Quantification of the protein expression levels of HMGCS2, PAD4, ME, and MPO in the above macrophages. **, P<0.01; ***, P<0.001; ****, P<0.0001 derived from one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Primary antibodies included: HMGCS2 (ab305,227; Abcam, USA), peptidyl arginine deiminase 4 (PAD4; 17,373-1-AP; Proteintech, China), macrophage elastase (ME; 22,989-1-AP; Proteintech), MPO (22,225-1-AP; Proteintech), GAPDH (T0004; Affinity, USA), β-Tubulin (66,240-1-Ig; Proteintech), and PPARα (66,826-1-Ig; Proteintech).

Techniques: Expressing, Control, Co-Immunoprecipitation Assay, Quantitative RT-PCR, Western Blot, Derivative Assay

Journal: Journal of Inflammation Research

Article Title: HMGCS2 Palmitoylation Promotes Diabetes-Induced Erectile Dysfunction Through PPARα-Mediated Macrophage Extracellular Trap Formation

doi: 10.2147/JIR.S623377

Figure Lengend Snippet: Pharmacological suppression of protein palmitoylation attenuates HG- and PA-induced ROS over-production, oxidative stress, and mitochondrial dysfunction in macrophages. ( A ) Representative intracellular ROS photographs by the use of DCFH-DA fluorescence probe in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. Scale bars, 50 μm. ( B ) Quantification of fluorescence intensity of ROS in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. ( C and D ) Measurement of MDA and GSH levels in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. ( E ) Representative JC-1 staining photographs in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. Green fluorescence represents that MMP is low and JC-1 exists in the cytoplasm in the form of monomer. Red fluorescence represents that MMP is high and JC-1 polymer is formed. Scale bars, 50 μm. ( F ) Calculation of a ratio of green fluorescence and red fluorescence for quantifying MMP changes. ( G ) Detection of ATP in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. ( H ) TEM for investigating the changes in mitochondrial morphology in control macrophages, HG- and PA-induced macrophages, and HG- and PA-induced macrophages with CMA treatment. Scale bars, 500 nm. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 derived from one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Primary antibodies included: HMGCS2 (ab305,227; Abcam, USA), peptidyl arginine deiminase 4 (PAD4; 17,373-1-AP; Proteintech, China), macrophage elastase (ME; 22,989-1-AP; Proteintech), MPO (22,225-1-AP; Proteintech), GAPDH (T0004; Affinity, USA), β-Tubulin (66,240-1-Ig; Proteintech), and PPARα (66,826-1-Ig; Proteintech).

Techniques: Fluorescence, Control, Staining, Polymer, Morphology, Derivative Assay

Journal: Journal of Inflammation Research

Article Title: HMGCS2 Palmitoylation Promotes Diabetes-Induced Erectile Dysfunction Through PPARα-Mediated Macrophage Extracellular Trap Formation

doi: 10.2147/JIR.S623377

Figure Lengend Snippet: Palmitoylation inhibitor CMA attenuates MET formation and macrophage infiltration in corpus cavernosum of DMED model rats. ( A-D ) Detection of levels of cfDNA, MPO, NE, and PAD4 in corpus cavernosum of control rats, DMED model rats, and DMED model rats with CMA administration. ( E ) Representative IHC staining of MPO, NE, and PAD4 in corpus cavernosum of control rats, DMED model rats, and DMED model rats with CMA administration. Scale bars, 50 μm. ( F–H ) Quantification of the protein expression levels of MPO, NE, and PAD4 in corpus cavernosum of control rats, DMED model rats, and DMED model rats with CMA administration. ( I ) Representative flow cytometry of CD68 + , CD11b + , and F4/80 + macrophages in corpus cavernosum of control rats, DMED model rats, and DMED model rats with CMA administration. ( J ) Quantification of the ratios of CD68 + , CD11b + , and F4/80 + macrophages in corpus cavernosum of control rats, DMED model rats, and DMED model rats with CMA administration. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001 derived from one-way ANOVA followed by Tukey’s post hoc test.

Article Snippet: Primary antibodies included: HMGCS2 (ab305,227; Abcam, USA), peptidyl arginine deiminase 4 (PAD4; 17,373-1-AP; Proteintech, China), macrophage elastase (ME; 22,989-1-AP; Proteintech), MPO (22,225-1-AP; Proteintech), GAPDH (T0004; Affinity, USA), β-Tubulin (66,240-1-Ig; Proteintech), and PPARα (66,826-1-Ig; Proteintech).

Techniques: Control, Immunohistochemistry, Expressing, Flow Cytometry, Derivative Assay

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: Genetic ablation of Lrg1 restrains tumor progression, normalizes vasculature, and reshapes the immune microenvironment. (A) Tumor growth curves, macroscopic anatomical images, and endpoint tumor weights of wild‐type (LRG1 +/+ ) and LRG1 knockout (LRG1 −/− ) subcutaneous MB49 tumor models. (B, C) Representative bioluminescence images (B) and macroscopic anatomical observations (C) of orthotopic bladder tumors in LRG1 +/+ and Lrg1 −/− mice. (D, E) Representative bioluminescence images with corresponding statistical analysis (D), and macroscopic anatomical images of the lungs detailing metastatic nodules along with their quantification (E) in the experimental tail‐vein injection model. (F) Immunofluorescence staining for CD31 and quantitative analysis of overall microvessel density and vessel diameter. (G, H) Representative confocal double immunofluorescence images for CD31 (green) and α‐SMA (red) (G) in subcutaneous tumor models and quantitative analysis of pericyte coverage (H) in both subcutaneous and orthotopic tumor models. (I) Scanning electron microscopy (SEM) images displaying the ultrastructure of tumor vascular endothelium and intraluminal membranous inclusions. (J) Expression profiles of canonical marker genes across different cell clusters, with red indicating high expression and blue indicating low expression. (K) Uniform Manifold Approximation and Projection (UMAP) plot displaying the distribution of major cell lineages in the mouse BCa tumor tissues, color‐coded by distinct clusters. (L) UMAP plot demonstrating the subclustering of neutrophils into four distinct subpopulations (C1‐C4). (M) UCell functional enrichment scoring of the NETs signature gene set within the neutrophil populations, where brighter colors represent higher enrichment scores. (N) Violin plots quantifying the NETs gene set scores across the neutrophil subpopulations. (O) Comparative relative proportions of the four neutrophil subpopulations between the LRG1‐OE and WT groups.

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: Knock-Out, Injection, Immunofluorescence, Staining, Electron Microscopy, Expressing, Marker, Functional Assay

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: Tumor‐derived LRG1 promotes cancer progression by recruiting neutrophils and driving NETosis. (A, B) Representative IHC images and quantification of CD66b+ neutrophil infiltration density in HM+ versus HM‐ BCa tissues. (C) Transwell assays evaluating the chemotactic effect of tumor‐derived LRG1 on isolated human neutrophils. (D) Representative macroscopic images and endpoint tumor weights demonstrating the “neutrophil‐dependent” tumor‐promoting effect of LRG1 in vivo, which is effectively rescued by anti‐Ly6G antibody depletion. (E) UMAP plot demonstrating the reclustering and annotation of distinct neutrophil subpopulations based on subcluster‐specific marker genes from the scRNA‐seq cohort. (F) Gene set enrichment scoring of the KEGG NETs pathway across neutrophil subpopulations, highlighting significant enrichment in the NCF2_neu subset. (G) Comparative analysis of the relative proportion of the NCF2_neu subpopulation between normal and BCa tumor tissues. (H) GSVA correlation analysis between the LRG1_tumor meta‐program and NCF2_neu infiltration abundance across three independent bulk RNA‐seq datasets. (I) ELISA quantification of serum NETs markers (cfDNA, MPO, NE) and their correlation with LRG1 levels. (J) Immunofluorescence staining for MPO and H3Cit in neutrophils directly stimulated with rh‐LRG1 or PMA. (K) Representative macroscopic images and endpoint tumor weights demonstrating the rescue of LRG1‐induced tumor progression by DNase I treatment in vivo.

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: Derivative Assay, Isolation, In Vivo, Marker, RNA Sequencing, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: NETs facilitate tumor cell trapping and directly destabilize tumor vasculature. (A) In vitro neutrophil adhesion assay evaluating the trapping effect of NETs on BCa cells and the reversal by DNase I. (B) Transwell co‐culture assay evaluating the impact of intact NETs on the migratory capacity of BCa cells. (C) In vitro tube formation assay utilizing HUVECs to assess the pro‐angiogenic capacity of NETs. (D) Endothelial permeability assay measuring TRITC‐Dextran flux across HUVEC monolayers following NETs stimulation. (E) Schematic timeline of the in vivo LPS‐induced systemic NETosis model and DNase I intervention. (F, G) Endpoint tumor weights (F) and macroscopic images (G) of subcutaneous tumors from respective groups. (H, I) Representative immunofluorescence images depicting neutrophil recruitment (Ly6G, H) and NETs formation (H3Cit, I) in tumor tissues. (J, K) Quantitative analysis of mural cell coverage (J) and representative confocal dual immunofluorescence images for CD31/α‐SMA (K) to confirm NET‐induced vascular destabilization.

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: In Vitro, Cell Adhesion Assay, Co-culture Assay, Tube Formation Assay, Permeability, In Vivo, Immunofluorescence

Journal: Advanced Science

Article Title: LRG1 Drives Pathological Angiogenesis by Disrupting Neutrophil Mitochondrial Homeostasis in Bladder Cancer

doi: 10.1002/advs.76604

Figure Lengend Snippet: LRG1 directly interacts with ANXA2 via its LRR domain and impedes the mitochondrial translocation of active Akt. (A) Silver staining and Venn diagram of mass spectrometry analysis identifying ANXA2 as a direct interactor following pull‐down assays with anti‐LRG1 or anti‐His antibodies. (B) Structural binding mode diagram illustrating the molecular interaction interface between LRG1 (yellow) and ANXA2 (blue). (C, D) Endogenous (C, neutrophils) and exogenous (D, HEK‐293T) Co‐IP assays validating the physical interaction between LRG1 and ANXA2. (E) Flow cytometric validation of CD11b expression in DMSO‐differentiated HL‐60 (dHL‐60) cells. (F) Confocal microscopy demonstrating the colocalization of LRG1 and ANXA2 in the cytoplasm of primary neutrophils and dHL‐60 cells. (G, H) Schematic of LRG1 truncation mutants (G) and Co‐IP assays (H) pinpointing the LRR1‐8 domain as essential for ANXA2 binding. (I) qRT‐PCR and Western blot analysis of ANXA2 expression following LRG1 stimulation. (J) Immunofluorescence confirming the colocalization of ANXA2 with the mitochondrial marker TOM20. (K, L) Immunoblotting of isolated mitochondrial fractions showing the suppressive effect of LRG1 on ANXA2 phosphorylation (K) and the concurrent reduction of mitochondrial pSer473‐Akt (L).

Article Snippet: Serum concentrations of LRG1 (RK01800, ABclonal, China), as well as the NETosis‐associated markers Myeloperoxidase (MPO; RK00310, ABclonal, China) and Neutrophil Elastase (NE; RK00694, ABclonal, China), were determined using commercial ELISA kits.

Techniques: Translocation Assay, Silver Staining, Mass Spectrometry, Binding Assay, Co-Immunoprecipitation Assay, Biomarker Discovery, Expressing, Confocal Microscopy, Quantitative RT-PCR, Western Blot, Immunofluorescence, Marker, Isolation, Phospho-proteomics