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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 <t>ATP</t> 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.
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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.

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: ATP content detection kit (HY-K0314-100 T, MCE) was employed for monitoring ATP.

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

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.

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: ATP content detection kit (HY-K0314-100 T, MCE) was employed for monitoring ATP.

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