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: Molecular Pain

Article Title: Nuclear factor kappa B regulated monocyte chemoattractant protein-1/chemokine CC motif receptor-2 expressing in spinal cord contributes to the maintenance of cancer-induced bone pain in rats

doi: 10.1177/1744806918788681

Figure Lengend Snippet: Intrathecal injection of CCR2 inhibitor (RS504393) reduced bone cancer pain and affected the production of inflammation factors. (a) CCR2 antagonist (RS504393) at a lower dose (50 μg) had mild effect on Walker-256 cell inoculation-induced pain hypersensitivity, whereas the antagonist at a higher dose (100 μg) reversed inoculation-induced mechanical allodynia for more than 4 h. *p < 0.05, **p < 0.01, vs. control serum. n = 12 rats per group. (b–d) CCR2 antagonist (RS504393) at a lower dose (25 μg) had a slight effect on inflammation cytokine and at a higher dose (50 μg) had a much more dramatic effect. *p < 0.05, **p < 0.01, vs. control serum. n = 4 rats per group. CIBP: cancer-induced bone pain; DMSO: dimethyl sulfoxide; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TNF-α: tumor necrosis factor-α; IL-4: interleukin-4; IFN-γ: interferon-γ.

Article Snippet: RS504393, an effective and selective antagonist of CCR2, was purchased from Boster (Shanghai, China).

Techniques: Injection, Control

Journal: Neuroscience bulletin

Article Title: Spinal CCL2 promotes central sensitization, long-term potentiation, and inflammatory pain via CCR2: Further insights into molecular, synaptic, and cellular mechanisms

doi: 10.1007/s12264-017-0106-5

Figure Lengend Snippet: Reversal of LTP of C-fiber-evoked field potentials in the dorsal horn of anesthetized mice by RS504393 (45 μg, 15 μL, i.t.), administered 2 h after LTP induction. Spinal LTP was induced by tetanic stimulation (100 Hz, 1 s, 4 trains) of C-fiber intensity in C57/B6 mice. *P < 0.05, vehicle (10% DMSO) vs RS504393, two-way ANOVA, n = 5 mice/group.

Article Snippet: Drugs and Administration RS504393, a potent and selective antagonist of CCR2, was from Tocris Bioscience (Bristol, UK).

Techniques:

Journal: Neuroscience bulletin

Article Title: Spinal CCL2 promotes central sensitization, long-term potentiation, and inflammatory pain via CCR2: Further insights into molecular, synaptic, and cellular mechanisms

doi: 10.1007/s12264-017-0106-5

Figure Lengend Snippet: Inhibition of CCL2- and CFA-induced heat hyperalgesia by intrathecal injection of RS504393. A Prevention of CCL2 (100 ng, i.t)-induced heat hyperalgesia by RS504393 (20 μg, i.t.). B Reversal of CFA-induced heat hyperalgesia by RS504393 (20 μg, i.t.) given 1 day after CFA injection. ***P < 0.001 vs vehicle control (10% DMSO), two-Way ANOVA followed by post-hoc Bonferroni test; n = 5–6 mice/group; data are expressed as mean ± SEM.

Article Snippet: Drugs and Administration RS504393, a potent and selective antagonist of CCR2, was from Tocris Bioscience (Bristol, UK).

Techniques: Inhibition, Injection, Control

Journal: Respiratory Research

Article Title: Loss of the adhesion G-protein coupled receptor ADGRF5 in mice induces airway inflammation and the expression of CCL2 in lung endothelial cells

doi: 10.1186/s12931-019-0973-6

Figure Lengend Snippet: Suppressive effect of a CCR2 antagonist on the expression of S100a8 , S100a9 , Slc26a4 , and Il5 in Adgrf5 −/− lungs. Two-week-old Adgrf5 −/− mice were administered RS504393 (2 mg/kg body weight) or vehicle once daily via subcutaneous injection for 8 days. The mRNA expression of Ccl2 , S100a8 , S100a9 , Saa3 , Slc26a4 , Clca1 , Tgfb1 , Il5 , and Il13 was analyzed by qPCR using total RNA isolated from post-lavage lungs of injected mice and non-injected WT mice at 3 weeks of age. The data were normalized to Gapdh levels and are expressed as values relative to those from corresponding vehicle-treated Adgrf5 −/− mice. Values are presented as the mean ± SEM ( n = 3–4). * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: RS504393 (Cayman Chemical, Ann Arbor, MI, USA) was dissolved in dimethylformamide at 10 mg/ml and then diluted to 0.4 mg/ml with a 0.9% sterile NaCl solution.

Techniques: Expressing, Injection, Isolation