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: bioRxiv

Article Title: Monocyte-derived macrophages promote intraepithelial infiltration of effector memory CD8 + T cells in tumors regressing after STING agonist treatment

doi: 10.64898/2026.05.28.728475

Figure Lengend Snippet: (A) Monocyte and macrophages diversity was assessed by spectral flow cytometry and divided into clusters, named after their markers expression signature (ncMo: Ly6C neg , CD43 + , cMo: Ly6C + CCR2 int , TAMo: Ly6C + CD64 + MHCII + , Immature TAM: CD64 int F4/80 int PDL1 + , TAM: CX3CR1 + F4/80 + CD64 + . (The undefined population did not fit in any of the previously detailed subpopulations). The tSNE represents the clusters present in control, day 4 and day 8 treated mice tumors and frequencies of each subpopulation for each condition are represented in color-coded pie charts. (B) Absolute numbers of monocytes (Ly6G neg Ly6C + ), macrophages (Ly6G neg Ly6C neg F4/80 + ) and monocyte-to-macrophage ratio were determined by flow cytometry in control (green) or day 4 treated tumors (purple). DMXAA, n=8 and controls n=6, from 3 independent experiments. Mann-Whitney statistical test. ** p < 0.01, **** p < 0.0001. (C) day 4 DMXAA, ratio % in MMTV-PyMT tumors. (D) IFNAR -/- PyMT tumors were transplanted in C56BL/6 mice and treated with DMXAA. Tumor volume is represented at day 0 and day 4 post-treatment, as well as (E) monocyte-to-macrophage ratio obtained through flow cytometry experiments at day 4. Controls n=5, DMXAA n=9 mice, from one experiment. (F) Monocyte-to-macrophage ratio represented against tumor volume foldchange (day 4 relative to day 0). Data is a merge from control (green) and treated (purple) mice, as well as from IFNAR -/- tumor experiments. Correlation analysis, r 2 =0.2384 with * p < 0.05.

Article Snippet: In the experiments with CCR2 inhibition, the antagonist (ref. RS504393 from MedChem Express) was added in 2 ml at the final concentration of 1 μM during this incubation time and the slices were rinsed before the staining step.

Techniques: Flow Cytometry, Expressing, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Monocyte-derived macrophages promote intraepithelial infiltration of effector memory CD8 + T cells in tumors regressing after STING agonist treatment

doi: 10.64898/2026.05.28.728475

Figure Lengend Snippet: (A) CCL2 production by control and day 4 treated tumors was assessed by dosing the supernatants of tumor slices after a 48 hours incubation. DMXAA, n= 4 and controls n= 5, from 2 independent experiments. T-test statistical test. ** p < 0.01. (B) The percentage of CD8+ T cells expressing CCR2 was determined by flow cytometry, in control and day 4 treated mice tumors. T-test statistical test was performed, ** p < 0.01. (C) Comparison of top 40 markers between cluster 4 of CD11b + from our work and Zhang’s study t-macro-CCL2 subset is represented in a Venn diagram for the number of intersect and individual genes. (D) Expression of the top 15 signature genes of t-macro-CCL2 (Zhang’s study) in CD11b + clusters from our work. The bubble plot allows for the representation of each gene average expression in cluster (bubble color) and the percentage of population expressing it (bubble size).

Article Snippet: In the experiments with CCR2 inhibition, the antagonist (ref. RS504393 from MedChem Express) was added in 2 ml at the final concentration of 1 μM during this incubation time and the slices were rinsed before the staining step.

Techniques: Control, Incubation, Expressing, Flow Cytometry, Comparison

Journal: Molecular Pain

Article Title: Acute and chronic phases of complex regional pain syndrome in mice are accompanied by distinct transcriptional changes in the spinal cord

doi: 10.1186/1744-8069-9-40

Figure Lengend Snippet: Amelioration of mechanical allodynia in CRPS mice following intrathecal, but not intraplantar, administration of the CCR2 antagonist, RS504393. Intrathecal RS504393 (3 μg) resulted in increased mechanical thresholds in the affected paw compared to vehicle-treated (*) and control mice (#). This was true for both the 3- (A) and 7-week (C) timepoints. Intraplantar injections failed to show any efficacy (B,D) . * p<0.05; ** p<0.01.n=8/group. Errors bars=S.E.M.

Article Snippet: The CCR2 antagonist, RS504393 (Santa Cruz Biotech, CA) was diluted in sterile saline containing DMSO (<1 %).

Techniques: Control

Journal: Chemokines

Article Title: Selective and Dual Targeting of CCR2 and CCR5 Receptors: A Current Overview

doi: 10.1007/7355_2014_40

Figure Lengend Snippet: CCR2 antagonists with spiropiperidine structure, inhibitory effects on CCL2 binding to human CCR2 receptor

Article Snippet: A prominent example of the spiropiperidine class of CCR2 inhibitors, RS504393 ( 12 ), was reported by Roche/Iconix.

Techniques: Binding Assay