Journal:

Article Title: Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

doi: 10.1073/pnas.0607514104

Figure Lengend Snippet: Chemokines in the LPS model of fetal loss. (A–E) Serum chemokine concentrations after LPS treatment in WT and D6−/− male mice. WT (open symbols) and D6−/− (filled symbols) mice were injected i.p. with 1.35 mg/kg LPS. At the indicated time points, circulating chemokine concentrations were measured by ELISA. Data are from seven mice for each time point. (F–J) Serum chemokine concentrations. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Circulating chemokine concentrations were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. (K–O) Chemokine levels in placenta. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Chemokine concentrations (expressed as nanograms of chemokine per milligram of total proteins of the lysates) were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. Results are reported as mean ± SEM. (A, F, and K) CCL22. (B, G, and L) CCL2. (C, H, and M) CCL11. (D, I, and N) CCL3. (E, J, and O) CXCL2.

Article Snippet: To block inflammatory chemokines, animals were treated with a mixture of goat antibodies to the mouse CC chemokines CCL3L1 (catalog no. AB450NA), CCL4 (catalog no. AB451NA), CCL2 (catalog no. AB479NA) and rat mAb anti-mouse CCL5 (catalog no. MAB478) purchased lyophilized from R&D Systems, resuspended in PBS, and mixed.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: In vitro citrullination of bacterially produced chemokines leads to their partial degradation. ( a ) Colloidal Coomassie stained SDS-PAGE analysis of the MCP-1/CCL2 products of in vitro citrullination. In vitro citrullination of commercially available recombinant MCP-1/CCL2 and self-made bacterially produced MCP-1/CCL2 performed with recombinant human PAD2 and PAD4 or rabbit PAD2, respectively. Colloidal Coomassie stained MCP-1 bands are shown while recombinant humanPAD2/4 or rabbit PAD2 (Sigma Aldrich/Merck, Waltham, MA, USA) were added to reactions into the quantities below the Colloidal Coomassie sensitivity limits and cannot be visualized. ( b ) Immunoblot analysis of bacterially produced MCP-1/CCL2 upon an in vitro citrullination reaction. Self-made full-length bacterially produced MCP-1/CCL2 was citrullinated in vitro with rabbit PAD2 and resolved on SDS-PAGE, stained with Ponceau S. detection of total MCP-1/CCL2 and modified citrullines with Senshu’s antibody that recognizes that modified citrullines were made according to Senshu’s protocol . Results shown are a representative of three or more repetitive experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: In Vitro, Produced, Staining, SDS Page, Recombinant, Western Blot, Modification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Mass-spectrometry verification of successful in vitro citrullination of MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 (mature form of the protein spans residues 24–99). The first row shows 23 amino acid-long signaling peptide and the second row–mature protein of 76 residues. All arginine residues are highlighted with circles. The boxed areas show the peptide that contains the citrullinated arginine residues that were detected. ( b ) Annotated tandem mass spectrometry (MS/MS) fragmentation spectrum for the citrullinated MCP1/CCL2 peptide, showing the citrullinated arginine residue at position 3 of the studied peptide (R45). The MS/MS fragmentation data were annotated using Expert System (Max Planck Institute of Biochemistry). The precursor ion was observed with a mass error of 1 part per million, and the error for the fragment ions was 0.02 daltons.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Mass Spectrometry, In Vitro, Sequencing, Tandem Mass Spectroscopy

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Detection of citrullinated recombinant human MCP-1/CCL2. Standard curves of citrullinated recombinant human MCP-1/CCL2 were set up using enzyme-linked immunosorbent assay in duplicate ( a ) standard curve of citrullinated E. coli produced recombinant human MCP-1/CCL2 chemokine (R&D Systems), ( b ) standard curve for citrullinated MCP-1/CCL2 chemokine produced by and purified from human HEK 293T cells. Absorbance at 450 nm is shown. Coefficients of determination R 2 are indicated in red on the relevant plots. Results shown are a representative of three or more repetitive experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Produced, Purification

Journal: International Journal of Molecular Sciences

Article Title: Mammalian Glycosylation Patterns Protect Citrullinated Chemokine MCP-1/CCL2 from Partial Degradation

doi: 10.3390/ijms24031862

Figure Lengend Snippet: Site-directed mutagenesis of potential glycosylation site at asparagine-14 in mammalian cell-produced MCP-1 significantly destabilizes recombinant human MCP-1/CCL2. ( a ) Amino acid sequence of MCP-1/CCL2 with indicated positions of predicted N-glycosylation and citrullination site confirmed by a mass-spectrometry. ( b ) Immunoblot analysis of bacterially versus mammalian cell-produced MCP-1/CCL2 upon an in vitro citrullination reaction. Bacterially produced wild-type MCP-1/CCL2 or HEK293T cell-produced wild-type and N14Q mutant version of chemokine were incubated with EDTA-free proteinase inhibitor cocktail supplemented cellular lysates prepared from the control (vehicle-transfected) or indicated hPAD enzyme-transfected HEK293T cells. Citrullination reactions that were performed directly within cellular lysates essentially as published before . Recombinant chemokine was concentrated with immunoprecipitation and resolved on 10–18% polyacrylamide gradient SDS-PAGE. Results shown are a representative of three experiments.

Article Snippet: Recombinant MCP-1/CCL2 duo-kit (ELISA) and monoclonal mouse antibody specific for MCP-1/CCL2 were purchased from R&D Systems (Minneapolis, MN, USA). pET19b vector was purchased from Novagen/Merck-Millipore (Utrecht, The Netherlands). pDEF vector was kindly provided by Dr. Goldman.

Techniques: Mutagenesis, Produced, Recombinant, Sequencing, Mass Spectrometry, Western Blot, In Vitro, Incubation, Transfection, Immunoprecipitation, SDS Page

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: CCL2 immunohistochemical staining in TCs and ICs. Upper row, ( A ) TCs, positive (ICs, negative); ( B ) Positive ICs with >6% CCL2 positivity (TCs, negative); Lower row, ( C ) TCs and ICs, negative; All photos are at 20× magnification.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Immunohistochemical staining, Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan–Meier analysis: Association of CCL2 staining in TCs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan–Meier analysis: Association between CCL2 expression in TCs and prognosis. Positive CCL2 expression in TCs was associated with a shorter mean OS ( p = 0.004), mean DSS ( p = 0.036), and mean RFS ( p = 0.047) than negative CCL2 expression.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Univariate and multivariate Cox’s regression analysis: Association of CCL2 staining in TCs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan–Meier analysis: Association of CCL2 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association between CCL2 expression in ICs and prognosis. Positive CCL2 expression in ICs was associated with a longer mean OS (P = 0.032), mean DSS (P = 0.001) and mean RFS (P = 0.001) than negative CCL2 expression.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Univariate and multivariate Cox’s regression analysis: Association of CCL2 staining in ICs with mean OS, mean DSS, or mean RFS.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression stratified by tumor stage (pT2 vs. pT3+4) in ICs and prognosis. Analysis of patients in the pT2 group revealed that positive CCL2 expression was associated with longer mean DSS ( p = 0.033) and mean RFS ( p = 0.022) than negative CCL2 expression. Comparably for patients in the pT3+4 group, CCL2 expression was associated with longer mean DSS ( p = 0.030) and mean RFS ( p = 0.034).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression stratified by lymph node stage; (N0 vs. N1+2) in ICs and prognosis. In the N0 group, CCL2-positive patients showed a longer mean OS ( p = 0.005), mean DSS and mean RFS (both p < 0.001) than CCL2-negative patients. However, in the N1+2 group, CCL2-positive patients had a shorter mean OS ( p = 0.001), mean DSS ( p = 0.031) and RFS ( p = 0.013).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression with ICs and prognosis in the patient group not treated with chemotherapy. In the chemotherapy untreated (CT–) group, CCL2 positivity was positively associated with OS ( p = 0.012), DSS ( p < 0.001) and RFS ( p < 0.001). However, there was no association between CCL2 staining and OS, DSS or RFS in the chemotherapy-treated group (CT+).

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Multivariate Cox’s regression analysis: Interactions of the parameters CCL2 staining, lymph node status, and application of chemotherapy.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Staining

Journal: Cancers

Article Title: CCL2 Expression in Tumor Cells and Tumor-Infiltrating Immune Cells Shows Divergent Prognostic Potential for Bladder Cancer Patients Depending on Lymph Node Stage

doi: 10.3390/cancers12051253

Figure Lengend Snippet: Kaplan-Meier analysis: Association of CCL2 expression in the basal subtype in ICs and prognosis. In the basal subtype, CCL2 positive staining was associated with a better mean DSS ( p = 0.032) and mean RFS ( p = 0.044) than CCL2 negative staining. However, no association between CCL2 staining and prognosis was found in the other molecular subtypes.

Article Snippet: Briefly, after heat pretreatment at 120 °C for 5 min with TE–buffer pH 9 and peroxidase blocking (Dako, Hamburg, Germany), primary antibody against CCL2 (monoclonal mouse IgG1, clone 2D8, Cat.-No. AM06749PU-N, dilution 1:15,000; Acris Antibodies, Herford, Germany) was applied for 30 min.

Techniques: Expressing, Staining, Negative Staining

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