Journal: Cell Biology and Toxicology

Article Title: SENP3 mediated DeSUMOylation of macrophage derived CCL17 accelerates atherosclerosis via regulation of Treg

doi: 10.1007/s10565-025-10099-3

Figure Lengend Snippet: Macrophages stimulated by oxLDL affect the chemotaxis of Treg through CCL17 and CCL22 competition. Macrophages were stimulated with oxLDL, and Treg cells were co-cultured with macrophage supernatant. A Immunofluorescence detection of co-localization of CCL22 and macrophages (CD68) in plaque tissue of AS mice. B The content of CCL22 in the supernatant of macrophages was detected by ELISA. C Transwell detected the effect of macrophages on the chemotaxis of Treg. ( D - E ) Transwell detected the migration of Treg cells. * p < 0.05, ** p < 0.01, *** p < 0.001. N = 3

Article Snippet: Prior to the transwell migration assay, the induced Tregs were treated with recombinant mouse CCL17 (MCE, HY-P71891A) or recombinant mouse CCL22 (MCE, HY-P7248) or anti-CCL17 (R&D, Catalog #: MAB529) and anti-CCL22 antibodies (R&D, Catalog #: MAB529).

Techniques: Chemotaxis Assay, Cell Culture, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Migration

Journal: Journal of Inflammation Research

Article Title: IRF3 Promotes Production of IL-6 and Nitric Oxide but Represses CCL22 in RAW264.7 Macrophage Cells Exposed to Lipopolysaccharides in Culture

doi: 10.2147/JIR.S496930

Figure Lengend Snippet: IRF3 deficiency in RAW264.7 cells enhances IL-6 and CCL22 production but decreases ISRE promoter activity and Nitric Oxide (NO) production in response to LPS. RAW-Lucia (wt RAW) or IRF3KO-RAW-Lucia cells (3KO RAW) at 5×10 5 per well incubated overnight were stimulated with 100 ng or 500 ng E. coli LPS (Ec-LPS) or P. gingivalis LPS (Pg-LPS) or stimulated with 10 μg/mL of poly (I)C (IC10) for 24 h at 37 °C after which secreted luciferase activity ( A ), IL-6 ( B and C ), Nitric Oxide (NO) ( D ) and CCL22 ( E and F ) production were evaluated. Bars represent mean ± standard error of mean (SEM) of quadruplicate samples from a representative experiment. * indicates significantly different from RAW Lucia response, p< 0.01, as determined by Student’s t -test.

Article Snippet: IL-6 concentrations in supernatants were determined using the mouse IL-6 ELISA kit obtained from ThermoFisher and CCL22 concentrations in supernatants using the mouse CCL22 ELISA kit of Peprotech (Cranbury, NJ).

Techniques: Activity Assay, Incubation, Luciferase

Journal: Journal of Inflammation Research

Article Title: IRF3 Promotes Production of IL-6 and Nitric Oxide but Represses CCL22 in RAW264.7 Macrophage Cells Exposed to Lipopolysaccharides in Culture

doi: 10.2147/JIR.S496930

Figure Lengend Snippet: P. gingivalis LPS significantly augments inflammatory cytokines from macrophages. RAW-Lucia cells (RAW cells) at 2×10 5 per well were incubated overnight and then stimulated with 500 ng E. coli LPS (Ec-LPS), 10 μg/mL ultrapure P. gingivalis LPS (uPg-LPS), 10 μg/mL of poly (I)C (IC), 50 ng/mL IFN-γ (IFN-g), or poly (I)C/IFN-γ (IC- IFN-g) in the presence or absence of 5 μg/mL conventional Pg-LPS (cPg-LPS) for 24 h at 37 °C. Then secreted luciferase activity ( A ), IL-6 ( B ), Nitric Oxide (NO) ( C ) and CCL22 ( D ) were evaluated. Bars represent mean ± standard error of mean (SEM) of quadruplicate samples from a representative experiment. * indicates significantly different from RAW Lucia response without cPg-LPS, p< 0.01, as determined by Student’s t -test.

Article Snippet: IL-6 concentrations in supernatants were determined using the mouse IL-6 ELISA kit obtained from ThermoFisher and CCL22 concentrations in supernatants using the mouse CCL22 ELISA kit of Peprotech (Cranbury, NJ).

Techniques: Incubation, Luciferase, Activity Assay

Journal: Journal of Inflammation Research

Article Title: IRF3 Promotes Production of IL-6 and Nitric Oxide but Represses CCL22 in RAW264.7 Macrophage Cells Exposed to Lipopolysaccharides in Culture

doi: 10.2147/JIR.S496930

Figure Lengend Snippet: P. gingivalis LPS significantly augments inflammatory cytokine responses from macrophages with IRF3 deficiency. IRF3KO-RAW-Lucia cells (3KO RAW) at 2×10 5 per well were incubated overnight and then stimulated with 500 ng E. coli LPS (Ec-LPS), 10 μg/mL ultrapure P. gingivalis LPS (uPg-LPS), 10 μg/mL of poly (I)C (IC), 50 ng/mL IFN-γ (IFN-g), or poly (I)C/IFN-γ (IC- IFN-g) in the presence or absence of 5 μg/mL conventional Pg-LPS (cPg-LPS) for 24 h at 37 °C. Then Relative ISRE promoter activity (secreted luciferase activity) ( A ), IL-6 ( B ), Nitric Oxide (NO) ( C ) and CCL22 ( D ) were evaluated. Bars represent mean ± standard error of mean (SEM) of quadruplicate samples from a representative experiment. * indicates significantly different from IRF3KO-RAW Lucia response without cPg-LPS, p< 0.01, as determined by Student’s t -test.

Article Snippet: IL-6 concentrations in supernatants were determined using the mouse IL-6 ELISA kit obtained from ThermoFisher and CCL22 concentrations in supernatants using the mouse CCL22 ELISA kit of Peprotech (Cranbury, NJ).

Techniques: Incubation, Activity Assay, Luciferase

Journal: Journal of Inflammation Research

Article Title: IRF3 Promotes Production of IL-6 and Nitric Oxide but Represses CCL22 in RAW264.7 Macrophage Cells Exposed to Lipopolysaccharides in Culture

doi: 10.2147/JIR.S496930

Figure Lengend Snippet: IRF3 augments inflammatory and diminishes anti-inflammatory responses from macrophages. IRF3KO-RAW-Lucia cells at 1×10 5 per well were incubated overnight and then transfected with an empty plasmid (pEGFP) or a mouse IRF3 expressing plasmid (pIRF3). After 24 h, transfected cells were stimulated with 500 ng E. coli LPS (Ec-LPS), 5 μg/mL P. gingivalis LPS (uPg-LPS), 10 μg/mL of poly (I)C (IC), 50 ng/mL IFN-γ (IFN-g), or poly (I)C/IFN-γ (IC- IFN-g) in the presence or absence of 5 μg/mL conventional Pg-LPS (cPg-LPS) for 24 h at 37 °C. Then Relative ISRE promoter activity (secreted luciferase activity) ( A ), IL-6 ( B ), Nitric Oxide (NO) ( C ) and CCL22 ( D ) were evaluated. Bars represent mean ± standard error of mean (SEM) of quadruplicate samples from a representative experiment. * indicates significantly different from IRF3KO-RAW Lucia response without cPg-LPS, p< 0.01, as determined by Student’s t -test.

Article Snippet: IL-6 concentrations in supernatants were determined using the mouse IL-6 ELISA kit obtained from ThermoFisher and CCL22 concentrations in supernatants using the mouse CCL22 ELISA kit of Peprotech (Cranbury, NJ).

Techniques: Incubation, Transfection, Plasmid Preparation, Expressing, Activity Assay, Luciferase