Journal: Journal of Translational Medicine

Article Title: N-acetyltransferase 10 affects the proliferation of intrahepatic cholangiocarcinoma and M2-type polarization of macrophages by regulating C-C motif chemokine ligand 2

doi: 10.1186/s12967-024-05664-z

Figure Lengend Snippet: NAT10 polarizes macrophages toward the M2 type through CCL2 . (A) Macrophages were polarized by treating them with the supernatant of ICC cells for 24 h. (B) After co-culturing ICC cells and macrophages for 24 h, the macrophages underwent polarization. (C) Co-culturing ICC cells with macrophages for 24 h resulted in the polarization of macrophages towards the M2 phenotype. (D) Immunofluorescence showed that CD86 expression increased and CD163 expression decreased in NAT10-knockdown tumors ( n = 6). Scale bars: 50 μm. (E and F) Western blot and ELISA showed that NAT10 knockdown decreased CCL2 expression levels in ICC cells and cell supernatant. (G) CCL2-knockdown cell lines were constructed and verified at the protein level. (H) Flow cytometry confirmed that CCL2 knockdown reduced the polarization of macrophages toward M2. (I) Immunofluorescence showed that CD86 expression increased and CD163 expression decreased in CCL2-knockdown tumors ( n = 6). Scale bars: 50 μm. Data are representative of three or more independent experimental replicates. Data are displayed as the mean ± SD. P -values were determined by Student’s t-test and one-way ANOVA in panels. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. ICC, intrahepatic cholangiocarcinoma; ELISA, enzyme-linked immunosorbent assay; SD, standard deviation; ANOVA, analysis of variance

Article Snippet: The levels of CCL2 in the cell supernatants were determined using an enzyme-linked immunosorbent assay (ELISA) kit (E-EL-H6005, Elabscience).

Techniques: Immunofluorescence, Expressing, Knockdown, Western Blot, Enzyme-linked Immunosorbent Assay, Construct, Flow Cytometry, Standard Deviation

Journal: Journal of Nanobiotechnology

Article Title: Folate-modified biomimetic nanovesicles loaded with a PU.1 inhibitor alleviate atherosclerosis by suppressing inflammation

doi: 10.1186/s12951-025-03825-w

Figure Lengend Snippet: PU.1 is upregulated in macrophages of advanced atherosclerotic lesions and promotes inflammation through IL-1β/NF-κB signaling. ( A ) Boxplot showing expression levels of PU.1 in early versus advanced atherosclerotic plaques based on the GSE43292 dataset. ( B ) UMAP plot of single-cell RNA sequencing data depicting major immune and stromal cell populations in atherosclerotic lesions. ( C ) UMAP feature plot showing SPI1 (encoding PU.1) expression predominantly enriched in macrophages. ( D ) Representative immunofluorescence staining of PU.1 (green) and CD68 (red) in aortic root sections from chow diet– and high fat diet–fed mice. Nuclei were counterstained with DAPI (blue). Scale bar: 200 μm. ( E ) CUT&Tag analysis showing genome-wide binding of PU.1 in macrophages. Heatmap indicates PU.1 enrichment near transcription start sites (TSS). ( F ) Genomic distribution of PU.1 binding peaks identified by CUT&Tag. ( G – H ) Representative CUT&Tag tracks showing PU.1 binding at the promoters of pro-inflammatory cytokines. ( I ) Dual-luciferase reporter assay confirming the transcriptional activation of the IL-1β promoter by PU.1 overexpression (OE). ( J ) Western blot showing that PU.1 knockdown suppressed ox-LDL–induced IL-1β expression and NF-κB pathway activation (p-IκB and p-p65). ( K ) Western blot demonstrating that IL-1β knockdown reversed PU.1-induced NF-κB activation. ( L ) Western blot analysis showing that the PU.1 inhibitor DB1976 attenuated ox-LDL–induced IL-1β expression and NF-κB activation. ( M ) qRT-PCR analysis of pro-inflammatory cytokines (IL-1β, IL-6, TNF-α, MCP-1) in macrophages treated with ox-LDL with or without DB1976. One-way ANOVA with Tukey’s multiple comparison post hoc test was used for statistical analysis. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: ELISA kits for mouse IL-1β (E-EL-M0037), IL-6 (E-EL-M0044), TNF-α (E-EL-M3063), and MCP-1 (E-EL-M3001) were purchased from Elabscience (China).

Techniques: Expressing, RNA Sequencing, Immunofluorescence, Staining, Genome Wide, Binding Assay, Luciferase, Reporter Assay, Activation Assay, Over Expression, Western Blot, Knockdown, Quantitative RT-PCR, Comparison, Standard Deviation

Journal: Journal of Nanobiotechnology

Article Title: Folate-modified biomimetic nanovesicles loaded with a PU.1 inhibitor alleviate atherosclerosis by suppressing inflammation

doi: 10.1186/s12951-025-03825-w

Figure Lengend Snippet: The anti-atherosclerotic effects of the D-FNVs. ( A - D ) BMDMs were co-treated with ox-LDL (100 µg/mL) and various formulations (free DB1976, D-NVs, D-FNVs). The levels of IL-1β, IL-6, TNF-α, and MCP-1 in the supernatant were measured by ELISA. ( E , F ) Flow cytometry analysis and quantification of intracellular ROS levels in BMDM cells treated with ox-LDL (100 µg/mL) and various formulations (free DB1976, D-NVs, D-FNVs), respectively, at 2 mM DB1976 for 24 h. ( G , H ) Flow cytometry analysis and quantification of apoptosis rates in BMDMs treated with ox-LDL (100 µg/mL) and various formulations (free DB1976, D-NVs, D-FNVs) at 2 mM DB1976 for 24 h. One-way ANOVA with Tukey’s multiple comparison post hoc test was used for statistical analysis. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: ELISA kits for mouse IL-1β (E-EL-M0037), IL-6 (E-EL-M0044), TNF-α (E-EL-M3063), and MCP-1 (E-EL-M3001) were purchased from Elabscience (China).

Techniques: Enzyme-linked Immunosorbent Assay, Flow Cytometry, Comparison, Standard Deviation

Journal: Journal of Nanobiotechnology

Article Title: Folate-modified biomimetic nanovesicles loaded with a PU.1 inhibitor alleviate atherosclerosis by suppressing inflammation

doi: 10.1186/s12951-025-03825-w

Figure Lengend Snippet: D-FNVs ameliorated inflammation in an atherosclerotic mouse model. ( A , B ) Representative images of plaques within the aortic root subjected to immunofluorescent staining for the macrophage marker CD68. Scale bar: 100 μm. ( C – F ) Levels of IL-1β, IL-6, TNF-α, and MCP-1 in aortic tissues collected from atherosclerotic mice treated with various formulations (saline, DB1976, D-NVs, D-FNVs). ( G – J ) Levels of IL-1β, IL-6, TNF-α, and MCP-1 in blood serum collected from the same groups of atherosclerotic mice. The n values are all biological replicates. One-way ANOVA with Tukey’s multiple comparison post hoc test was used for statistical analysis. Data are presented as the mean ± standard deviation (SD). * p < 0.05, ** p < 0.01, and *** p < 0.001. **** p < 0.0001

Article Snippet: ELISA kits for mouse IL-1β (E-EL-M0037), IL-6 (E-EL-M0044), TNF-α (E-EL-M3063), and MCP-1 (E-EL-M3001) were purchased from Elabscience (China).

Techniques: Staining, Marker, Saline, Comparison, Standard Deviation

Journal: Frontiers in immunology

Article Title: Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

doi: 10.3389/fimmu.2021.638676

Figure Lengend Snippet: FIGURE 1 | DcR3.Fc- or HBD.Fc-treated M-Mφ and GM-Mφ secreted low amount of interleukin-1β (IL-1β) under particle stimulation. Six-day-cultured M-Mφ (A,C) and GM-Mφ (B) were derived from wild-type (WT) B6 and cocultured with hIgG (3 µg/ml), DcR3.Fc (3 µg/ml), or HBD.Fc (3 µg/ml). Cells were primed with lipopolysaccharides (LPS; 100 ng/ml) for 4 h and treated with monosodium urate (MSU) crystal, alum, or silica (150 or 300 µg/ml) for 6 h or ATP (3 mM) for 1 h. The concentration of IL-1β was measured by ELISA. All data shown were mean ± SD from three independent experiments. The statistical significance was determined by one-way ANOVA. *p < 0.05, **p < 0.01, and ***p < 0.001 were obtained by comparing the DcR3.Fc- or HBD.Fc-treated group to the hIgG-group. “NS” means no statistical significance.

Article Snippet: MouseM-CSF, mouse GM-CSF, human caspase-1, andmouse IL1β, IL-6, CCL2, CXCL2, and CXCL1 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Cell Culture, Derivative Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in immunology

Article Title: Decoy Receptor 3 Inhibits Monosodium Urate-Induced NLRP3 Inflammasome Activation via Reduction of Reactive Oxygen Species Production and Lysosomal Rupture.

doi: 10.3389/fimmu.2021.638676

Figure Lengend Snippet: FIGURE 2 | DcR3 suppressed MSU-induced caspase-1 activation but did not affect the expression of NLRP3 or pro-IL-1β in LPS-treated macrophages. M-Mφ (A,C) and GM-Mφ (B,C) were treated with hIgG, DcR3.Fc, or HBD.Fc (3 µg/ml for each) for 6 days during differentiation stage in M-Mφ and GM-Mφ. Cells were treated with LPS (100 ng/ml) for 4 h, and total RNA was extracted. The mRNA levels of pro-IL-1β and NLRP3 were measured by real-time PCR (A,B). In some experiments, after LPS priming, cells were treated with MSU (300 µg/ml) for 3 h. The supernatants were harvested for caspase 1 p10 ELISA analysis (C). Data indicated mean ± SD of three independent experiments. The statistical significance was determined by one-way ANOVA. *p < 0.05 and ***p < 0.001 were obtained by comparing DcR3.Fc- or HBD.Fc-treated group to hIgG-group.

Article Snippet: MouseM-CSF, mouse GM-CSF, human caspase-1, andmouse IL1β, IL-6, CCL2, CXCL2, and CXCL1 ELISA kits were purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: Shared pathways significantly upregulated in endothelial cells treated with EVs.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Protein-Protein interactions, Migration, Chemotaxis Assay, Variant Assay, Activity Assay, Activation Assay

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: Endothelial cells were left untreated or treated for 16 hrs with LPS (1μg/mL) or EVs derived from non-infected or Mtb -infected macrophages. (A) Cells were stained with FITC conjugated anti-mouse TLR2 antibody or FITC conjugated anti-mouse IgG1 antibody as an isotype control. (B) Cells were surface-stained with FITC-labeled rat anti-mouse VCAM1 or FITC labeled anti-rat IgG2a antibody as an isotype control. (C) Cells were permeabilized and stained for intracellular CCL2 using PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gates were set to approximately 1% for isotype control and were maintained for all subsequent analysis. RC: untreated cells, RvEV: Treatment with EVs from H37Rv-infected macrophages, UnEV: Treated with EVs from non-infected macrophages. Data is representative of the protein expression from three independent experiments.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Derivative Assay, Infection, Staining, Control, Labeling, Expressing

Journal: PLoS ONE

Article Title: Activation of endothelial cells by extracellular vesicles derived from Mycobacterium tuberculosis infected macrophages or mice

doi: 10.1371/journal.pone.0198337

Figure Lengend Snippet: (A) SVEC4-10 cell monolayers were left untreated or stimulated for 3 hrs with EVs derived from non-infected or Mtb -infected mice. CFSE-labeled mouse BMMs were added to SVEC4-10 cells. The fluorescently-labeled macrophages which migrated through the SVEC4-10 cell monolayer into the bottom of the Transwell filter were imaged. The number of BMMs in seven randomly selected fields were counted and the total number of cells for each condition defined. The data is the average of three independent mouse Mtb infections +SD with (*) indicating a p value < 0.05 compared to RC. (B) Quantitative RT-PCR was performed on endothelial cells that were left untreated or treated for 4 hours with EVs derived from uninfected or Mtb -infected macrophages. Total RNA was extracted followed by cDNA synthesis. Fold change of gene expression was calculated by comparative Ct method. Data is from two independent mouse Mtb infections. (C) Scatter plots of flow cytometry analysis of CCL2 expression. Endothelial cells were left untreated or treated for 16 hours with EVs derived from non-infected or Mtb -infected macrophages. Permeabilized cells were stained with PE-conjugated anti-mouse CCL2 antibody or PE-labeled IgG as an isotype control. Gate was set for isotype control and was maintained for all subsequent analysis. RC: untreated cells. Un-EV: serum-derived EVs from uninfected mice, D7-EV, D14-EV, D21-EV: serum derived EVs from mice infected for 7, 14 and 21 days respectively. Data is representative of the CCL2 expression from two independent experiments.

Article Snippet: For CCL2 antibody staining cells were treated with 2μM monensin solution (BioLegend) for 2hrs after EV treatment to block protein secretion from the cell.

Techniques: Derivative Assay, Infection, Labeling, Quantitative RT-PCR, cDNA Synthesis, Gene Expression, Flow Cytometry, Expressing, Staining, Control

Journal: Molecular medicine reports

Article Title: 5‑aza‑2'‑deoxycytidine promotes migration of acute monocytic leukemia cells via activation of CCL2‑CCR2‑ERK signaling pathway.

doi: 10.3892/mmr.2017.6737

Figure Lengend Snippet: Figure 3. 5‑Aza treatment increased the expression of CCL2 and CCR2. (A) THP‑1 cells were treated with DMSO or 0.5 µM 5‑Aza for 72 h, and CCR2 and CCL2 expression levels were analyzed by reverse transcription‑polymerase chain reaction. (B) Western blot analysis of CCR2 expression. (C) Enzyme‑linked immunosorbent assay analysis of CCL2 levels in the culture medium. Data are presented as the mean ± standard error of the mean (n=4). **P<0.01 vs. DMSO. 5‑Aza, 5‑aza‑2'‑deoxycytidine; CCL2, C chemokine (C‑C motif) ligand 2; CCR2, C‑C chemokine receptor type 2; DMSO, dimethyl sulfoxide.

Article Snippet: The chemokine (C-C motif) ligand 2 (CCL2) enzyme-linked immunosorbent assay (ELISA) kit (catalog no. DCP00) was obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: Molecular medicine reports

Article Title: 5‑aza‑2'‑deoxycytidine promotes migration of acute monocytic leukemia cells via activation of CCL2‑CCR2‑ERK signaling pathway.

doi: 10.3892/mmr.2017.6737

Figure Lengend Snippet: Figure 4. Inhibition of CCL2‑CCR2 signaling reverses the 5‑Aza‑induced increase in THP‑1 cell migration. THP‑1 cells were treated with 0.5 µM 5‑Aza for 72 h, and a cell migration assay was performed in the absence or presence of 10 µM RS. Data are presented as the mean ± standard error of the mean (n=4). *P<0.001 vs. DMSO and #P<0.001 vs. 5‑Aza. CCL2, chemo kine (C‑C motif) ligand 2; CCR2, C‑C chemokine receptor type 2; 5‑Aza, 5‑aza‑2'‑deoxycytidine; RS, RS102895; DMSO, dimethyl sulfoxide.

Article Snippet: The chemokine (C-C motif) ligand 2 (CCL2) enzyme-linked immunosorbent assay (ELISA) kit (catalog no. DCP00) was obtained from R&D Systems, Inc. (Minneapolis, MN, USA).

Techniques: Inhibition, Migration, Cell Migration Assay