Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: We measured the relative changes in MCP-1, MMP-1 , MMP-9, and TIMP gene expression by real-time PCR. Data are presented as the fold change in gene expression normalized to the endogenous reference gene PDHB and relative to untreated controls. Panel 1 , the effect of 10 μM concentration of CCR2 RS504393 inhibiting compound was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. CCR2 RS504393 inhibiting compound was diluted with DMSO and dispensed in 5 μl volume to produce a final culture concentration of 0.01% DMSO. We also added 5 μl of DMSO to control cultures. The results presented are from six independent experiments showing the mean and standard deviations. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.01) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. Notably, levels of specific mRNAs from non-stimulated cells were not significantly different than those obtained from cultures that proceeded with the CCR2 inhibitor alone. Panel 2 , the effect of 1 μM concentration of MMP-1 inhibitor 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide was assessed following 24 hr in vitro stimulation of THP-1 cells with 5 μg/ml sonicated H37Rv M. tuberculosis . Cultures proceeded in 500 μl serum-free RPMI. 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide was diluted in incomplete RPMI. The results presented are from six independent experiments showing the mean and standard deviations from the mean. We consistently observed significant differences in the mean values across variables (Kruskal-Wallis p < 0.01) when testing MCP-1, MMP-1 and MMP-9. We show corrected p-values obtained from student t-tests. Of note, levels of specific mRNAs from non-stimulated cells were not significantly different from those obtained from cultures that proceeded with the 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide inhibitor alone. The inhibitors’ concentrations were selected from dose response-experiments.

Article Snippet: THP-1 cells (1×10 6 cells/ml) were stimulated with the indicated amounts of H37Rv M. tuberculosis lysate obtained after sonication (sonicated H37Rv M. tuberculosis ) as described previously (Ganachari et al. 2010), or the indicated amounts of CCR2 RS504393 (Tocris), MMP-1 4-Aminobenzoyl-Gly-ProD-Leu-D-Ala hydroxamic peptide (SIGMA), PAR-1 SCH79797 (Tocris) inhibitors, and human purified MMP-1 (hMMP-1) activated with p-aminophenylmercuric acetate (EMD Chemicals).

Techniques: Gene Expression, Real-time Polymerase Chain Reaction, Concentration Assay, In Vitro, Sonication, Control

Journal: Genes and immunity

Article Title: Host gene-encoded severe lung TB: from genes to potential pathways

doi: 10.1038/gene.2012.39

Figure Lengend Snippet: We used three-color FACS analysis for these experiments. Exposure of quiescent THP-1 cells to sonicated H37Rv M. tuberculosis for 24 hr induced the differentiation of these cells into CD14-positive/CD16-negative cells. In Panels 1 and 2, Section A shows a low proportion of CD14-positive/CD16-negative cells in quiescent THP-1 cells; Section B shows an increment in the proportion of CD14-positive/CD16-negative THP-1 cells in response to sonicated H37RV M. tuberculosis exposure.; Section C shows minimal (non-significant) variation in this response to sonicated H37Rv M. tuberculosis exposure in the presence of the MMP-1 inhibitor (Panel 1) or CCR2 inhibitor (Panel 2). In Sections D, E, and F of Panel 1, we show that the presence of MMP-1 inhibitor 4-Aminobenzoyl-Gly-Pro-D-Leu-D-Ala hydroxamic peptide prevents the down-regulation of PAR-1 expression by THP-1 cells exposed to sonicated H37Rv M. tuberculosis exposure. In Sections D, E, and F of Panel 2, we show that the presence of CCR2 inhibitor RS504393 also prevents the down-regulation of PAR-1 expression by THP-1 cells exposed to sonicated H37Rv M. tuberculosis exposure. We acquired 100,000 events for experiments in Panel 1, while we acquired 10,000 events for experiments in Panel 2, according to the number of live cells gathered in each experiment. Of note, the MMP-1 inhibitor was dissolved in incomplete RPMI while the CCR2 inhibitor was dissolved in DMSO to obtain a DMSO final culture concentration of 0.01%. Three experiments were done for the assessment of each inhibitor.

Article Snippet: THP-1 cells (1×10 6 cells/ml) were stimulated with the indicated amounts of H37Rv M. tuberculosis lysate obtained after sonication (sonicated H37Rv M. tuberculosis ) as described previously (Ganachari et al. 2010), or the indicated amounts of CCR2 RS504393 (Tocris), MMP-1 4-Aminobenzoyl-Gly-ProD-Leu-D-Ala hydroxamic peptide (SIGMA), PAR-1 SCH79797 (Tocris) inhibitors, and human purified MMP-1 (hMMP-1) activated with p-aminophenylmercuric acetate (EMD Chemicals).

Techniques: Sonication, Expressing, Concentration Assay

Journal: Cell reports

Article Title: Bone Marrow Mesenchymal Stromal Cell-Derived Periostin Promotes B-ALL Progression by Modulating CCL2 in Leukemia Cells.

doi: 10.1016/j.celrep.2019.01.034

Figure Lengend Snippet: Figure 6. CCL2 Promotes the Expression of POSTN in BM-MSCs via STAT3 Activation (A) qRT-PCR analysis of the levels of POSTN, c-JUN, RUNX1, RUNX2, STAT3, and YY1 in BM-MSCs and rmCCL2-treated BM-MSCs (n = 3 per group). (B) Western blot analysis of the levels of POSTN, phosphorylated STAT3 (p-STAT3), and STAT3 in BM-MSCs isolated from mice with or without B-ALL. (C) Western blot analysis of the levels of POSTN, p-STAT3, and STAT3 in BM-MSCs and rmCCL2-treated BM-MSCs pre-incubated with or without RS504393. (D) Western blot analysis of the levels of p-STAT3 and STAT3 in BM-MSCs and rmCCL2-treated BM-MSCs pre-incubated with or without RS504393. (E) Western blot analysis of the levels of POSTN, p-STAT3, and STAT3 in BM-MSCs and co-cultured BM-MSCs pre-incubated with or without RS504393. (F) Western blot analysis of the levels of p-STAT3 and STAT3 in mouse BM-MSCs co-cultured with L1210-GFP-Luc-sh-ctrl cells or L1210-GFP-Luc-sh-CCL2 cells. (G) Western blot analysis of the levels of p-STAT3 and STAT3 in BM-MSCs transfected with siRNAs to knock down STAT3. (H and I) qRT-PCR assay of the mRNA levels of STAT3 (H) and POSTN (I) in mouse BM-MSCs and rmCCL2-treated and siRNA-transfected BM-MSCs (n = 3 per group). (J) Western blot analysis of the levels of POSTN, p-STAT3, and STAT3 in mouse BM-MSCs and rmCCL2-treated and siRNA-transfected BM-MSCs. (K) Analysis of the effect of STAT3 knockdown in BM-MSCs on CCL2-promoted adhesion of L1210-GFP-Luc cells to mouse BM-MSCs (n = 6 per group). The graphs show mean ± SD. Significance is indicated as follows: **p < 0.01; ***p < 0.001; ns, no significance.

Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Mouse monoclonal anti-POSTN Adipogen Cat# AG-20B-0033 Rabbit monoclonal anti-GFP Abcam Cat# ab183734 Mouse monoclonal anti-MCP-1 Cell Signaling Technology Cat# 2029 Rabbit monoclonal anti-Intergrin Linked ILK Abcam Cat# ab52480 Rabbit monoclonal anti-Phospho-NF-kB p65 Cell Signaling Technology Cat# 3033T Rabbit monoclonal anti-NF-kB p65 Cell Signaling Technology Cat# 8242 Rabbit monoclonal anti-Phospho-STAT3 Cell Signaling Technology Cat# 9145 Mouse monoclonal anti-STAT3 Cell Signaling Technology Cat# 9132 Rabbit monoclonal anti-AKT Cell Signaling Technology Cat# 9272 Rabbit monoclonal anti-Phospho-FAK Cell Signaling Technology Cat# 3281 Mouse monoclonal anti-Phospho-JNK Cell Signaling Technology Cat# 9255 Rabbit monoclonal anti-JNK Cell Signaling Technology Cat# 9252 Rabbit monoclonal anti-Phospho-AKT Cell Signaling Technology Cat# 9271 Anti-Integrin aVb3 antibody Millipore/Merck Cat# MAB1876-Z Anti-Integrin aVb5 antibody Millipore/Merck Cat# MAB1961 Rabbit monoclonal anti-Ki67 Abcam Cat# ab15580 Rabbit monoclonal anti-ERK Cell Signaling Technology Cat# 4695 Rabbit monoclonal anti-Phospho-ERK Cell Signaling Technology Cat# 3211 Mouse monoclonal anti-FAK BD Biosciences Cat# 610087 Rabbit monoclonal anti-b-actin ABclonal Cat# AC026 Goat anti-Rabbit IgG (H+L) Secondary Antibody, HRP Thermo Fisher Scientific Cat# 31460 Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP Thermo Fisher Scientific Cat# 31430 Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 Thermo Fisher Scientific Cat# A10040 Biological Samples Human bone marrow tissue samples The First Affiliated Hospital of Hunan Normal University N/A Human serum samples The First Affiliated Hospital of Hunan Normal University N/A Chemicals, Peptides, and Recombinant Proteins CCR2 Chemokine receptor anagonist RS504393 TOCRIS Cat# 2517 ILK inhibitor Cpd 22 Millipore/Merck Cat# 407331 BAY11-7082 MedChemExpress Cat# HY-13453 PD98059 MedChemExpress Cat# HY-12028 Human Periostin/OSF-2 DuoSet ELISA R&D Cat# DY3548B Human CCL2/MCP-1 DuoSet ELISA R&D Cat# DY279 Recombinant Human Periostin/OSF-2 Protein, CF R&D Cat# 3548-F2 Recombinant Mouse Periostin/OSF-2 Protein, CF R&D Cat# 2955-F2 Recombinant Mouse CCL2/JE/MCP-1 Protein R&D Cat# 479-JE-050 (Continued on next page) e1 Cell Reports 26, 1533–1543.e1–e4, February 5, 2019

Techniques: Expressing, Activation Assay, Quantitative RT-PCR, Western Blot, Isolation, Incubation, Cell Culture, Transfection, Knockdown

Journal: Cell reports

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation

doi: 10.1016/j.celrep.2024.114193

Figure Lengend Snippet: (A) Conditioned medium (CM) was collected from primary WT and Slc4a4 KO astrocytes and subjected to LC-MS/MS-based unbiased proteomics and cytokine/chemokine array. (B) Angiogenic factors detected from LC-MS/MS-based unbiased proteomics. (C) Cytokine/chemokines changed in the CM from WT and Slc4a4 KO astrocytes. Pooled CM from three independent cultures per genotype were used (D) CCL2 level was measured by ELISA in CM collected under either normal or oxygen-glucose-deprivation (OGD) condition cortices from uninjured and stroked (1 dpi) brains from WT and Slc4a4-icKO mice. Each dot represents CM or cortices collected from an individual animal. n = 3–5 per genotype. * p < 0.05, ** p < 0.01 by Student’s t test. In vivo astrocytic CCL2 expression after stroke at 4 dpi was quantified from double immunostaining. n = 17–21 cells collected from 3–5 mice per genotype. * p < 0.05 by Student’s t test. (E) Representative images of astrocytic CCL2 expression at 4 dpi by double immunostaining of CCL2/GFAP. (F and G) Endothelial CCR2 mRNA expression was visualized by RNA scope and CD31 staining and quantified by counts of CCR2+ puncta. Each dot represents an individual blood vessel. n = 49 vessels collected from 4 mice per genotype. **** p < 0.0001 by Student’s t test. (H and I) Representative images and quantification of endothelial CCR2 expression from double immunostaining. Each dot represents an individual animal. n = 4–5 per genotype. * p < 0.05 by Student’s t test. (J) Proposed model for the Slc4a4-CCL2-CCR2 axis regulating astrocyte-endothelial interaction. All data are presented as mean ± SEM.

Article Snippet: CCR2 antagonist , Tocris , 2517/10.

Techniques: Liquid Chromatography with Mass Spectroscopy, Enzyme-linked Immunosorbent Assay, In Vivo, Expressing, Double Immunostaining, RNAscope, Staining

Journal: Cell reports

Article Title: Astrocytic Slc4a4 regulates blood-brain barrier integrity in healthy and stroke brains via a CCL2-CCR2 pathway and NO dysregulation

doi: 10.1016/j.celrep.2024.114193

Figure Lengend Snippet:

Article Snippet: CCR2 antagonist , Tocris , 2517/10.

Techniques: Purification, Virus, Recombinant, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Colorimetric Assay, Mutagenesis, Software, Microscopy

Journal: Advanced Science

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen‐Mediated Chemokine Secretion in Pancreatic Cancer

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Collagens modulate chemokine expression in PDAC cells under IFNγ stimulation. A) Tumor growth of sgCtrl and sgCcn1 KPC cells inoculated subcutaneously in C57B/L6 mice. Tumor‐bearing mice were treated with CCR2 inhibitor RS504393 and CXCR2 inhibitor SB225002. n = 6 mice per group. B) Representative images of tumors (left) and quantification of tumor weight (right) in mice inoculated with sgCtrl or sgCcn1 KPC cells. Tumor‐bearing mice were treated with the CCR2 inhibitor RS504393 and the CXCR2 inhibitor SB225002. C) Flow cytometric analysis of MDSC proportions in subcutaneous sgCtrl and sgCcn1 KPC tumors. D) mRNA levels of collagens, including Col4a1, Cola4a2, Col5a1, Col6a1, Col6a2, Col6a3, and Col8a1, in KPC cells after their total knockdown. E) mRNA levels of chemokines, including Ccl2, Ccl20, Cxcl1, Cxcl3, Cxcl5, Csf1, Csf2, and Csf 3 , in KPC cells after their total knockdown. F–H) mRNA levels of chemokines, including Ccl2, Ccl7, Ccl20, Cxcl1, Cxcl3, Cxcl5, Csf1, Csf2, and Csf3, in KPC cells after Col5a1 knockdown (F), Col6a3 knockdown (G), and Col8a1 knockdown (H). I) mRNA levels of collagens, including Col4a1, Cola4a2, Col5a1, Col6a1, Col6a2, Col6a3, and Col8a1, in sgCtrl KPC cells after treatment with 100 ng mL −1 IFNγ. J) mRNA levels of Col5a1, Col6a3, and Col8a1 measured in siCol5a1, siCol6a3, and siCol8a1 KPC cells after treatment with 100 ng mL −1 IFNγ. K–M) mRNA levels of chemokines including the Ccls (K), Cxcls (L), and Csfs (M), in siCol5a1, siCol6a3, and siCol8a1 KPC cells after treatment with 100 ng mL −1 IFNγ. N) mRNA level of Il6 in siCol5a1, siCol6a3, and siCol8a1 KPC cells after treatment with 100 ng mL −1 IFNγ.

Article Snippet: CCR2 inhibitor RS504393 (S9738, Selleck), CXCR2 inhibitor SB225002 (S7651, Selleck), and gemcitabine (S1714, Selleck) were purchased from Selleckchem.

Techniques: Expressing, Knockdown

Journal: Advanced Science

Article Title: CCN1 Enhances Tumor Immunosuppression through Collagen‐Mediated Chemokine Secretion in Pancreatic Cancer

doi: 10.1002/advs.202500589

Figure Lengend Snippet: Ccn1 regulates the TME in PDAC. A) Schematic representation of macrophage isolation from mouse bone marrow. B) Macrophage invasion evaluated by the chemotactic effect of sgCtrl and sgCcn1 KPC cells in a Transwell invasion assay. C) Macrophage polarization assessed by flow cytometry in macrophages cocultured with sgCtrl and sgCcn1 KPC cells. D) Macrophage polarization assessed by flow cytometry in macrophages cocultured with sgCtrl and sgCcn1 KPC cells after treatment with CCR2 inhibitor RS504393 and CXCR2 inhibitor SB225002. E) Survival of sgCtrl and sgCcn1 KPC cells cocultured with macrophages, assessed by CCK‐8 assay. F) Cell death of sgCtrl and sgCcn1 KPC cells cocultured with macrophages, assessed by flow cytometry. G) Protein expression levels of TNFα in macrophages cocultured with sgCtrl and sgCcn1 KPC cells. H) mRNA expression levels of TNFα in macrophages cocultured with sgCtrl and sgCcn1 KPC cells. I) Protein expression level of c‐Caspase 3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 TNFα. J) Protein expression level of c‐Caspase 3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 TNFα, mouse CCN1 protein, and cilengitide (αVβ3 and αVβ5 integrin inhibitor). K) Nucleus localization of CCN1 in KPC cells with CCN1 (red) and DAPI (blue) fluorescence. L) Protein expression level of p‐STAT3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 INFγ. M) mRNA levels of Ccl7, Cxcl3, and Csf3 in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 INFγ. N) mRNA level of Il6 measured in sgCtrl and sgCcn1 KPC cells after treatment with 100 ng mL −1 INFγ.

Article Snippet: CCR2 inhibitor RS504393 (S9738, Selleck), CXCR2 inhibitor SB225002 (S7651, Selleck), and gemcitabine (S1714, Selleck) were purchased from Selleckchem.

Techniques: Isolation, Transwell Invasion Assay, Flow Cytometry, CCK-8 Assay, Expressing, Fluorescence