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