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