Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 increases cell surface expression of P2X4R protein in microglial cells. P2X4R protein on the surface of primary cultured microglial cells was detected by cell surface biotinylation followed by Western blot analysis of P2X4R protein using its specific antibody. Total cellular P2X4R protein was detected in whole-cell lysates of microglial cells. Microglial cells in primary cultures were treated with either CCL2 (100 ng/mL), CX3CL1 (100 ng/mL), or IFN-γ (100 U/mL) for 30 min (a) with CCL2 (10, 30, and 100 ng/mL) for 30 min (b) and with CCL2 (100 ng/mL) for 30, 60, and 120 min (c). b, c histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four to five times, and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05 compared with unstimulated control microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCR2 mediates CCL2-induced surface P2X4R expression. a–c Microglial cells in primary cultures were pretreated with either CCL2 neutralizing antibodies (CCL2-Ab, 2.5 and 25 μg/mL) for 30 min (a), RS-504393 (RS, 1 and 10 μM) for 20 min (b) or forskolin (10 μM) for 10 min (c) before CCL2 (100 ng/mL) stimulation. d Microglial cells were stimulated by CCL12 (10 ng/mL) for 30 min. Surface proteins of microglial cells were biotinylated 30 min after CCL2 (a–c) and CCL12 (d) stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four to five times, and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, **P < 0.01, ***P < 0.001 compared with unstimulated control microglia; #P < 0.05, ##P < 0.01 compared with CCL2-stimulated microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 is involved in fibronectin-induced surface P2X4R upregulation. a Biotinylation assay of surface P2X4R proteins. CCL2 neutralizing antibody (CCL2-Ab, 25 μg/mL) and normal goat IgG (IgG, 25 μg/mL) were pretreated for 30 min before fibronectin (FN, 10 μg/mL) stimulation. Surface proteins of microglial cells were biotinylated 24 h after fibronectin stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated five times and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, **P <0.01 compared with unstimulated control microglia; #P < 0.05 compared with fibronectin-stimulated microglia. b CCL2 levels in supernatants of microglial cultures were measured by ELISA; primary cultured microglial cells were treated with or without fibronectin (10 μg/mL) for various time points of testing. The experiments were repeated six times, and the data are means ± SEM of the concentration of CCL2 (pg/mL). ***P < 0.001 compared with control at 1 h

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Cell Surface Biotinylation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 enhances movement of P2X4Rs. a Immunocytochemical analysis of P2X4Rs detected by its specific antibody. Primary cultured microglia were stimulated by CCL2 (100 ng/mL) for 30 min and were fixed by formaldehyde. Cells were pretreated with CCL2 neutralizing antibody (CCL2-Ab, 25 μg/mL) and forskolin (10 μM) for 30 and 10 min, respectively, before CCL2 stimulation. b Monitoring of P2X4R-GFP fluorescence particles in living microglial cells transduced with a lentiviral vector encoding P2X4R-GFP. Left Typical trajectories of P2X4R-GFP fluorescence particles in microglia treated with HBSS (control) or CCL2 (100 ng/mL). Right The total distance (μm, means ± SEM, n = 19) of the trajectories of P2X4R-GFP fluorescence particles from initial position. ***P < 0.001 compared with the control group by two-way ANOVA. Scale bar: 10 μm

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Cell Culture, Fluorescence, Transduction, Plasmid Preparation, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: Dynamin inhibitor does not affect the effect of CCL2 on cell surface P2X4R expression. Microglial cells in primary cultures were pretreated with the dynamin inhibitor dynasore (80 μM) for 30 min before CCL2 (100 ng/mL) stimulation. Surface proteins of microglial cells were biotinylated 30 min after CCL2 stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four times and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, ***P < 0.001 compared with unstimulated control microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 causes lysosomal exocytosis in microglial cells. a Immunofluorescence for P2X4R (green) and LGP85 (red), a marker of lysosome, in primary cultured microglial cells treated with or without CCL2 (100 ng/mL) for 30 min. b The release of the lysosomal enzyme β-hexosaminidase from microglial cells was assayed by measuring its activity in the supernatants of microglial cultures collected at 30 min after CCL2 (100 ng/mL) or ionomycin (5 μM) stimulation. The experiments were repeated six times and the data are means ± SEM. *P < 0.05, **P < 0.01 compared with unstimulated control microglia. Scale bar: 10 μm

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Immunofluorescence, Marker, Cell Culture, Activity Assay, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 enhances P2X4R-mediated Akt phosphorylation in microglial cells. Primary cultured microglia were treated with or without CCL2 (100 ng/mL) for 30 min. Western blot analysis of the levels of phosphorylated and total Akt protein in whole-cell lysate of CCL2-treated microglia 10 min after ATP (5 μM) stimulation. Cells were pretreated with TNP-ATP (100 μM) 5 min before ATP stimulation. A histogram of the relative band density ratio of Akt phosphorylation normalized to the levels of β-actin. The experiments were repeated five times, and the data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Phospho-proteomics, Cell Culture, Western Blot

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CD54⁺ iCAFs promote monocyte migration and M2-like polarization through CCL2 secretion. A Left: Transwell migration assay of THP-1 cells co-cultured with CD54⁺ iCAFs or normal fibroblasts (NFs) for 48 h. Right: Quantification of migrated cells (n = 3 independent experiments). Scale bar: 100 μm. B-C THP-1 cells co-cultured with CD54⁺ iCAFs show upregulated expression of M2-like macrophage biomarkers and cytokines. D CD54⁺ iCAFs were transfected with CD54-targeting siRNA (si-CD54) or negative control siRNA (si-NC). Volcano plot of differentially expressed genes (DEGs) identified by RNA-seq analysis between si-CD54 and si-NC groups. E CD54 + iCAFs were transfected with si-CD54 or si-NC. Then, CD54 and CCL2 protein levels were assessed by Western blot. Left: Representative images. Right: Quantification from n = 3 independent experiments. F ELISA quantification of CCL2 secretion from CD54⁺ iCAFs and NFs. G Left: THP-1 cell migration in response to recombinant CCL2 treatment (50 ng/ml, 48 h) or PBS (Control group). Right: Quantification of migrated cells. All quantitative data are presented as mean ± SD. Statistical significance was determined using Student’s t-test for panels A (right), B, C, E (right), F, and G (right). *: P < 0.05, **: P < 0.01, ***: P < 0.001; ns, not significant

Article Snippet: A neutralizing anti-human CCL2 antibody (MedChemExpress) or an IgG isotype control was introduced to the culture medium.

Techniques: Migration, Transwell Migration Assay, Cell Culture, Expressing, Transfection, Negative Control, RNA Sequencing, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Control

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CD54⁺ iCAF-driven macrophage reprogramming promotes CXCL8 expression and tumor progression in vivo. A THP-1-derived macrophages, transfected with control siRNA (Control group) or ITGAL-targeting siRNA (Treatment group), were co-cultured with CD54⁺ iCAFs for 48 h and subsequently subjected to RNA-seq analysis. Volcano plot shows differentially expressed genes (DEGs) between control and treatment groups. B Left: Representative flow cytometry plots of CD54⁺ iCAF abundance. Middle: Quantitative analysis of CXCL8⁺ cell frequencies in CD54⁺ iCAF-high tumors (n = 3). Right: quantitative analysis of CXCL8 expression in CD54 + iCAF-high versus CD54 + iCAF-low (Displaying in Supplementary Fig. 5F) tumors. C Flow cytometric quantification of CXCL8-producing immune cell subsets in cervical cancer tissues. D Multiplex immunohistochemistry images showing macrophage-specific CXCL8 expression in cervical cancer tissue (Scale bar: 100 μm). E NIH/3T3 fibroblasts transfected with CD54 overexpression plasmid (OE-CD54) or empty vector control (OE-NC). CD54 and CCL2 secretion levels were quantified by ELISA. F Representative tumor images from C57BL/6 mice co-injected with TC-1 cells and NIH/3T3 fibroblasts expressing CD54 or empty vector (Control). G Tumor growth curves measured every 3 days (n = 3 mice/group). H-I Flow cytometry analysis of CD206⁺ macrophage infiltration in tumors from (F). I (Right): Quantification of CD206⁺ macrophage proportions. J Serum MIP-2 levels measured by ELISA in experimental groups from (F). All quantitative data are presented as mean ± SD. Statistical significance was determined using Student’s t-test for panels B (right), E, G(right), I (right), and J. *: P < 0.05, **: P < 0.001, ***: P <0.001

Article Snippet: A neutralizing anti-human CCL2 antibody (MedChemExpress) or an IgG isotype control was introduced to the culture medium.

Techniques: Expressing, In Vivo, Derivative Assay, Transfection, Control, Cell Culture, RNA Sequencing, Flow Cytometry, Multiplex Assay, Immunohistochemistry, Over Expression, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Injection

Journal: Molecular Cancer

Article Title: Integrated multi-omics identifies a CD54 + iCAF-ITGAL + macrophage niche driving immunosuppression via CXCL8-PDL1 axis in cervical cancer

doi: 10.1186/s12943-025-02471-y

Figure Lengend Snippet: CXCL8 correlates with CD8⁺ T cell exclusion and promotes PD-L1 expression on macrophages through cell-contact and soluble-factor dependent mechanisms. A CIBERSORT analysis showing an inverse correlation between CXCL8 mRNA levels and CD8⁺ T cell infiltration. B Representative immunohistochemistry (IHC) images showing CD8⁺ T cell density in high- versus low-CXCL8 expressing tumors (Scale bar: 100 μm). C Negative correlation between protein levels of CXCL8 and PD-L1 based on IHC scoring (Pearson correlation). D Left: Flow cytometry plots of PD-L1⁺ cells. Right: Quantification of PD-L1 expression in high- versus low-CXCL8 tumors (n = 3 per group). E Representative IHC staining confirming macrophage-specific PD-L1 expression (Scale bar: 100 μm). F Left: Gating strategy for identifying PD-L1⁺ cells. Right: Quantification of PD-L1 expression across immune cell subtypes, showing macrophage dominance (n = 6). G Left: Flow cytometry profiles of PD-L1 expression. Right: PD-L1 levels in high- versus low-CXCL8 tumors. H Flow cytometry analysis of CXCL8 and PD-L1 expression on CD68+ macrophages co-cultured with CD54⁺ iCAFs under direct contact or Transwell conditions, with normal fibroblasts (NFs) and macrophage-only cultures as controls. The bar graph shows geometric mean fluorescence intensity from three independent experiments. Corresponding representative flow cytometry plots are shown in Supplementary Fig. 6D. I CXCL8 and PD-L1 expression on macrophages after co-culture with CD54⁺ iCAFs and treatment with IgG control, anti-CD54, or anti-ITGAL blocking antibodies. See Supplementary Fig. 6E for flow plots. J CXCL8 and PD-L1 expression on macrophages transfected with control siRNA (si-NC) or ITGAL-targeting siRNA (si-ITGAL), with or without CD54 + iCAF co-culture. Corresponding representative flow cytometry plots are shown in Supplementary Fig. 6F. K CXCL8 and PD-L1 expression on macrophages co-cultured with CD54⁺ iCAFs and treated with IgG control or anti-CCL2 neutralizing antibody. Representative flow plots are provided in Supplementary Fig. 6G. Data are presented as mean ± SD. Statistical tests used: two-tailed Student’s t-test (D, right; G, right; K); one-way ANOVA with Tukey's multiple comparisons test (F, right; H, I, J). **: P < 0.01, ***: P < 0.001; ns, not significant

Article Snippet: A neutralizing anti-human CCL2 antibody (MedChemExpress) or an IgG isotype control was introduced to the culture medium.

Techniques: Expressing, Immunohistochemistry, Flow Cytometry, Cell Culture, Fluorescence, Co-Culture Assay, Control, Blocking Assay, Transfection, Two Tailed Test