Journal: International Journal of Molecular Sciences

Article Title: Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation

doi: 10.3390/ijms222212480

Figure Lengend Snippet: Effect of statin treatment on inflammatory macrophage activation. ( A ) RAW-Blue TM cells were treated with either simvastatin (Sim, 2 µM) or cerivastatin (Cer, 1 µM) for 24 h. Inflammatory activation was induced by treatment with LPS (100 ng/mL) for the final 4 h. NF-κB/AP-1 activity was determined by secreted embryonic alkaline phosphatase (SEAP) detection. Co = solvent control ( n = 3, triplicates). ( B , D , F , H ) Tnf ( B ), Il1b ( D ), Il6 ( F ), and Nos2 ( H ) mRNA expression in BMMs was determined by real-time RT-PCR, normalized to Ppia , and expressed as x-fold of Co. BMMs were stimulated for the last 4 h with LPS (100 ng/mL) or polarized towards M1 (LPS, 100 ng/mL; IFNγ, 20 ng/mL), or M2 (IL4, 20 ng/mL) in the presence or absence of Sim (2 µM) or Cer (0.5 µM) for 24 h. Co = solvent control ( n = 6). ( C , E ) TNF ( C ) and IL1β ( E ) were measured by bioassay. BMMs were treated for 24 h with either Sim (2 µM) or Cer (0.5 µM). Inflammatory activation was induced by treatment with LPS (100 ng/mL for IL1β, 10 ng/mL for TNF) for the final 4 h. Co = solvent control ( n = 3, duplicates for TNF, triplicates for IL1β). ( G ) Nitrite production was measured by Griess assay. BMMs were treated for 24 h with either Sim (2 µM) or Cer (0.5 µM). Samples were stimulated for the final 20 h (LPS, 50 ng/mL; IFNγ, 20 ng/mL). Co = solvent control ( n = 3, triplicates). A one-sample t -test followed by a Bonholm post hoc test was used for analyzing gene expression data of the control group. Means of more than two groups were compared by one-way ANOVA with Bonholm post hoc test (normal distribution). * p < 0.05, ** p < 0.01, and *** p < 0.001.

Article Snippet: Murine M-CSF (#130-101-704), IFNγ (#130-105-782), IL4 (#130-094-061), and Il1β (#130-101-681) were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Activation Assay, Activity Assay, Solvent, Control, Expressing, Quantitative RT-PCR, Bioassay, Griess Assay, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation

doi: 10.3390/ijms222212480

Figure Lengend Snippet: Statins affect different signaling pathways in macrophages. ( A ) Intracellular cholesterol levels. BMMs were either treated for 24 h with simvastatin (Sim, 2 µM) or cerivastatin (Cer, 0.5 µM). Co = solvent control ( n = 3, triplicates). ( B ) RAW-Blue TM cells were treated for 24 h with either Sim (2 µM) or Cer (1 µM). Cells were co-treated with mevalonate (MVA, 100 µM) where indicated. Inflammatory activation was induced by treatment with LPS (100 ng/mL) for the final 4 h. NF-κB/AP-1 activity was determined by secreted embryonic alkaline phosphatase (SEAP) detection. Co = solvent control ( n = 3, triplicates). ( C ) IL1β was measured by bioassay. BMMs of Nlrp3 WT and KO BMMs were treated for 24 h with either Sim (2 µM) or Cer (0.5 µM). Inflammatory activation was induced by treatment with LPS (100 ng/mL) for the final 4 h. Co = solvent control ( n = 4 each WT and KO, quadruplicates). ( D ) mRNA expression of indicated genes were determined by real-time RT-PCR, normalized to Ppia , and expressed as x-fold of Co. BMMs were either treated for 24 h with Sim (2 µM) or Cer (0.5 µM). Co = solvent control ( n = 6). ( E , F ) ERK phosphorylation was examined by Western Blot analysis. BMMs were treated with Sim (2 µM) or Cer (0.5 µM) for one hour. Co = solvent control. ( E ) One representative blot is shown. ( F ) Signal intensities were quantified and normalized to total ERK ( n = 3). ( G ) RAW-Blue TM cells were pre-treated for 30 min with the ERK inhibitor PD98059 (Inh, 10 µM). Cells were treated for 24 h with either Sim (2 µM) or Cer (1 µM). Inflammatory activation was induced by treatment with LPS (100 ng/mL) for the final 4 h. NF-κB/AP-1 activity was determined by secreted embryonic alkaline phosphatase (SEAP) detection. Co = solvent control ( n = 3, triplicates). One-sample t -test followed by Bonholm post hoc test was used for analyzing gene expression data of the control group and Western blot ( D , F ). Means of more than two groups were compared by one-way ANOVA with Bonholm post hoc test (normal distribution) ( B , C , G ). * p < 0.05, ** p < 0.01.

Article Snippet: Murine M-CSF (#130-101-704), IFNγ (#130-105-782), IL4 (#130-094-061), and Il1β (#130-101-681) were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Protein-Protein interactions, Solvent, Control, Activation Assay, Activity Assay, Bioassay, Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, Gene Expression

Journal: International Journal of Molecular Sciences

Article Title: Statins and Bempedoic Acid: Different Actions of Cholesterol Inhibitors on Macrophage Activation

doi: 10.3390/ijms222212480

Figure Lengend Snippet: Effect of bempedoic acid treatment macrophages. ( A ) RAW-Blue TM cells were treated with bempedoic acid (Bemp, 25 µM) for 24 h. Inflammatory activation was induced by treatment with LPS (100 ng/mL) for the final 4 h. NF-κB/AP-1 activity was determined by secreted embryonic alkaline phosphatase (SEAP) detection. Co = solvent control ( n = 3, triplicates). ( B , D , F ) Tnf ( B ), Il1b ( D ), and Il6 ( F ), mRNA expression in BMMs was determined by real-time RT-PCR, normalized to Ppia , and expressed as x-fold of Co. BMMs were stimulated for the last 4 h with LPS (100 ng/mL) or polarized towards M1 (LPS, 100 ng/mL; IFNγ, 20 ng/mL) or M2 (IL4, 20 ng/mL) in the presence or absence of Bemp (25 µM) for 24 h. Co = solvent control ( n = 6). ( C , E ) TNF ( C ) and IL1β ( E ) were measured by bioassay. BMMs were treated for 24 h with Bemp (25 µM). Inflammatory activation was induced by treatment with LPS (100 ng/mL for IL1β, 10 ng/mL for TNF) for the final 4 h. Co = solvent control ( n = 3, duplicates for TNF, triplicates for IL1β). ( G , H ) BMMs were treated for 24 h with Bemp (25 µM) in the presence or absence of IL4 (20 ng/mL) and monitored by an IncuCyte S3 system after the addition of fluorogenic pHrodo ® Red S. aureus bioparticles (5 µg/well). Quantification of phagocytotic activity expressed as mean red fluorescence intensity normalized to confluence ( n = 4, duplicates). RFU = relative fluorescence units. A one-sample t-test followed by a Bonholm post hoc test was used for analyzing gene expression data of the control group. Means of more than two groups were compared by one-way ANOVA with Bonholm post hoc test (normal distribution). Statistical analysis of phagocytotic activity was performed by two-way ANOVA with Bonholm post hoc test. *** p < 0.001.

Article Snippet: Murine M-CSF (#130-101-704), IFNγ (#130-105-782), IL4 (#130-094-061), and Il1β (#130-101-681) were obtained from Miltenyi Biotech (Bergisch Gladbach, Germany).

Techniques: Activation Assay, Activity Assay, Solvent, Control, Expressing, Quantitative RT-PCR, Bioassay, Fluorescence, Gene Expression

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic diagram of mice feeding and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . b–n Male AMPKα fl/fl mice and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 6 weeks were fed HFD with or without IL-1β neutralizing antibody (1 mg/kg, twice one week) to explore the obesity development. Immunofluorescent staining of IL-1β in BAT and ScWAT ( b ), body weight gain ( c , n = 5 mice), relative fat and lean mass ( d , n = 5 mice), the weight of Liver ( e , n = 5 mice), BAT ( f , n = 5 mice), and ScWAT ( g , n = 5 mice), representative H&E staining of the liver, BAT and ScWAT ( h ), insulin tolerance test ( i , n = 5 mice), the rectal temperature in cold exposure at 4 °C for different times ( j , k , n = 5 mice), immunohistochemical staining of UCP-1 in BAT ( l ), the proinflammatory genes of ScWAT ( m , Il1b, Tnfa, Nos2, Ccl2 and F4/80: n = 5 mice in each group, Il6: n = 4 mice in LysM-Cre, AMPKα fl/fl + IL-1β mAb group and n = 5 mice in other group), and the immunohistochemical staining of F4/80 in BAT and ScWAT ( n ). Data are presented as the mean ± SEM, groups were compared by two-way ANOVA followed by Fisher’s LSD test ( c – g , i – k , m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a–t Male LysM-Cre, IL-1β fl/fl mice, LysM-Cre, IL-1β fl/fl , AMPKα fl/fl mice, AMPKα fl/fl mice, IL-1β fl/fl mice, and LysM-Cre, AMPKα fl/fl mice with C57BL/6 background at the age of 8 weeks were fed HFD to explore the obesity and related phenotype. Body weight change ( a , n = 8 mice), body weight gain ( b , n = 8 mice), representative mice image ( c ), relative fat and lean mass ( d , n = 8 mice), the representative image of liver, BAT and ScWAT ( e ), the metabolic organ weight of liver ( f , n = 8 mice), BAT ( g , n = 8 mice) and ScWAT ( h , n = 8 mice), representative H&E staining of liver, BAT and ScWAT ( i ), insulin tolerance test ( j , n = 8 mice), the rectal temperature in cold exposure at 4 °C for different times ( k , l , n = 8 mice), immunohistochemical staining of UCP-1 in BAT ( m ), the proinflammatory genes of ScWAT ( n – s , n = 8 mice) and immunohistochemical staining of F4/80 in BAT and ScWAT ( t ). Data are presented as the mean ± SEM, groups were compared by one-way ANOVA followed by Fisher’s LSD test ( b , d , f – h , right of j , l , n – s ) or two-way ANOVA followed by Fisher’s LSD test ( a ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Immunohistochemical staining

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Schematic representation of the potential substrate of AMPK in succinate formation, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 . Co-immunoprecipitation analysis of the interaction of AMPKα with SUCLA2 ( b ) or SUCLG2 ( c ) in RAW 264.7 cells. d Immunoblot analysis of indicated proteins in BMDMs that are transfected with siRNA for 30 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. e Immunoblot analysis of indicated proteins in AMPKα fl/fl BMDMs (Flox) and LysM-Cre, AMPKα fl/fl BMDMs (MKO) that are transfected with siRNA for 18 h followed by stimulation with 100 ng/mL LPS for an additional 12 h. f Immunoblot analysis of the expression of SUCLG1 and SUCLA2 after AMPKα was knocked down by siRNA for 48 h in RAW 264.7 cells. g The relative enzymatic activity of SUCLA2 (the direction from succinyl-CoA to succinate) was detected after AMPKα was knocked down by siRNA for 36 h followed by stimulation with 100 ng/mL LPS for an additional 12 h in RAW264.7 cells ( n = 3 biological replicates). h In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. i Venn diagram was used to integrate the phosphorylation site that was detected by mass spectrometry and predicted by GPS 5.0 ( http://gps.biocuckoo.cn/ ) or HPRD . j Protein sequence alignment indicated the conservation of SUCLA2 at Ser60 across multiple species. k Immunoblot analysis of indicated proteins in the in vitro kinase assay that mixed the purified His-CAMKKβ, His-AMPKα1β1γ1 with His-SUCLA2 (WT) or His-SUCLA2 (S60A). l Immunoblot analysis of indicated proteins in BMDMs after incubation with 200 µM A-769662 for 3 h. m In vitro phosphorylation analysis was performed by mixing purified His-CAMKKβ, His-AMPKα1β1γ1 and His-SUCLA2 (WT) or His-SUCLA2 (S60A) in the presence of ATP-g-S, and immunoblot analysis of indicated proteins with indicated antibodies. The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2 ( n ) or S60A-SUCLA2 ( o ) after incubation with AMPK in vitro (n = 3 biological replicates). p The enzymatic activity (the direction from succinyl-CoA to succinate) of purified WT-SUCLA2, S60A-SUCLA2, and S60D-SUCLA2 in the same protein content (n = 4 biological replicates). q–r The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2, S60A-SUCLA2 and S60D-SUCLA2 through lentivirus infection for 48 h followed the treatment by 100 ng/mL LPS for 6 h in the condition of glutamine replete ( q ) or deprived ( r ) condition (n = 4 biological replicates). The relative gene expression of IL-1β in RAW264.7 cells that overexpress human WT-SUCLA2 with S60A-SUCLA2 ( s ) or S60D-SUCLA2 ( t ) through lentivirus infection for 48 h, the cells were treated with 200 µM A-769662 for 3 h in advance followed by 100 ng/mL LPS for 6 h (n = 4 biological replicates). Data are presented as the mean ± SEM, groups were compared by the unpaired two-tailed Student’s t test (g) or one-way ANOVA followed by Bonferroni’s multiple-comparisons test ( q , r ) or two-way ANOVA followed by Bonferroni’s multiple-comparisons test ( n , o , p , s , t ), representative data are shown from one of the three independent experiments ( b – f , h , k – m ). P < 0.05 was considered to be statistically significant.

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Immunoprecipitation, Western Blot, Transfection, Expressing, Activity Assay, In Vitro, Phospho-proteomics, Purification, Mass Spectrometry, Sequencing, Kinase Assay, Incubation, Gene Expression, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Macrophage SUCLA2 coupled glutaminolysis manipulates obesity through AMPK

doi: 10.1038/s41467-025-57044-w

Figure Lengend Snippet: a Immunofluorescent staining of CD68, pT 172 -AMPK, pS 60 -SUCLA2 and IL-1β in the subcutaneous adipose tissue from lean (BMI = 21.5), overweight (BMI = 25.7) or obese (BMI = 32.0) subjects, representative data are shown from one of the three independent experiments. b Model of how macrophage AMPK responds to glutaminolysis and regulates glutaminolysis-coupled IL-1β expression, and this image was created in BioRender. Zang, Y. (2025) https://BioRender.com/e16k510 .

Article Snippet: To trigger the activation of the inflammasome and detect secreted IL-1β, macrophages were exposed to 2 mM ATP (#HY-B2176, MCE) for 30 min after the incubation with 100 ng/mL LPS, and the secreted IL-1β was detected by mouse IL-1β Elisa kit (#abs520001, Absin).

Techniques: Staining, Expressing