ccr2 inhibitor  (MedChemExpress)

Journal: Journal of Advanced Research

Article Title: Novel function of macrophage migration inhibitory factor in regulating post-infarct inflammation and the therapeutic significance

doi: 10.1016/j.jare.2025.05.030

Figure Lengend Snippet: Macrophage migration inhibitory factor (MIF) promotes inflammatory cell migration and infiltration through C – C motif chemokine receptor 2 (CCR2) and C-X-C motif chemokine receptor (CXCR) 4 and promotes splenic monocyte mobilization via interaction with angiotensin Ⅱ type 1 receptor (AT-1R) after myocardial infarction (MI). A, peripheral blood mononuclear cells (PBMCs) chemotaxis in response to homogenized sham or infarct heart tissue from wild type (WT) mice at day 3 post-MI, or MI homogenate together with inhibition of C–C motif chemokine receptor 2 (CCR2), C-X-C motif chemokine receptor (CXCR) 2 and CXCR4, respectively. Cells/HPF, cells/high power field. PBMCs harvested from WT mice at 3 days post-MI. CCR2i, CCR2 inhibition. CXCR2i, CXCR2 inhibition. CXCR4i, CXCR4 inhibition. n = 4–8 per group. *** P < 0.001 vs. sham . B, PBMCs chemotaxis in response to recombinant human MIF (rMIF) or rMIF with inhibition of CCR2, CXCR2 and CXCR4, respectively. PBMCs harvested from WT mice at 3 days post-MI. n = 4–8 per group. *** P < 0.001 vs. control . C, Representative immunoblotting images for monocyte chemoattractant protein-1 (MCP-1), CXCR4 and internal reference protein (heat shock protein 60, HSP-60) in hearts from WT and global MIF deficient (MIFKO) mice with sham-operation or 72 h MI. D-E, Quantitative analysis of MCP-1 and CXCR4 expression. n = 6–9 per group. ** P < 0.01 vs. sham in the same genotype. *** P < 0.001 vs. sham in the same genotype. F, Splenic monocyte (from normal WT mice) chemotaxis in response to plasma from normal mice or mice with 1.5 h MI, or MI plasma together with losartan. Lor, losartan, AT-1R inhibitor. n = 6–8 per group. *** P < 0.001 vs. normal. G, Splenic monocytes (from normal WT mice) chemotaxis in response to rMIF and angiotensin II (Ang II), or together with losartan addition, respectively. n = 6–8 per group. ** P < 0.01 vs. Control. *** P < 0.001 vs. Control. H, Representative immunoblotting images for AT-1R and internal reference protein (HSP-60) in spleens from WT and global MIF deficient (MIFKO) mice with sham-operation and 1.5 h MI. I, Quantitative analysis of AT-1R expression. n = 8 per group. *** P < 0.001 vs. sham in the same genotype . J. Co-immunoprecipitation (Co-IP) assay using an anti-AT-1R antibody or IgG demonstrated the interaction between AT-1R and MIF, as well as between AT-1R and CD74 in splenic monocytes and spleen tissue. Input, input sample. IgG, negative control sample. IP, Co-IP sample.

Article Snippet: For both groups 1 and 2, PBMCs were pre-treated for 1 h using DMEM with or without CCR2 inhibitor (CCR2 antagonist 4 hydrochloride, 100 mM), CXCR2 inhibitor (SB225002, 800 nM) or CXCR4 inhibitor (Plerixafor, AMD3100, 10 μM) (all from MedChemExpress, USA).

Techniques: Migration, Chemotaxis Assay, Inhibition, Recombinant, Control, Western Blot, Expressing, Clinical Proteomics, Co-Immunoprecipitation Assay, Negative Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Piezo1 activation suppresses bone marrow adipogenesis to prevent osteoporosis by inhibiting a mechanoinflammatory autocrine loop

doi: 10.1038/s41392-025-02455-w

Figure Lengend Snippet: Piezo1 suppresses Lcn2 expression and secretion via the inhibition of Ccl2-evoked NF-κB activation. BMMSCs were isolated from femurs and tibias of 10-week-old male PDGFRα-Piezo1 KO mice and WT controls. a Gene expression of Ccr2 in BMMSCs, as determined by real-time PCR. b–g Recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) was added to the adipogenic and osteogenic induction medium during BMMSC differentiation. The expression levels of osteogenic genes ( b–d ) and adipogenic genes ( e–g ) were determined via real-time qPCR. h–n WT and KO BMMSCs were treated with rmCcl2 (100 ng/mL) or the CCR2 antagonist INCB3344 (10 nM) for 24 h. h Sonicated BMMSCs were subjected to chromatin immunoprecipitation (ChIP) using an anti-p65 antibody. The ChIP DNA samples were then subjected to qPCR amplification using specific primers against the promoter region of Lcn2, as shown in supplementary Fig. . Representative gel images of end-point ChIP-qPCR products are shown. i Quantitative results are shown as the percentage of input DNA: % of Input = 2^(CT Input −CT IP )/dilution factor × 100%. j Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Lcn2 promoter (Lcn2-WT) or the mutated Lcn2 promoter with mutations in the NF-κB binding motif (Lcn2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. k Schematic diagram illustrating the actions of rmCcl2, the CCR2 antagonist, and the NF-κB-specific inhibitor QNZ on NF-κB translocation and Lcn2 expression; created with BioRender ( https://BioRender.com ). l ELISA analysis of total p65 in the nucleus and cytoplasm of BMMSCs. m Representative immunofluorescence images of intracellular p65 localization. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. n P65 nuclear translocation was quantified by ImageJ software via Pearson’s correlation coefficient (PCC) between the p65 immunofluorescence signal and DAPI staining across segmented nuclear regions. o , p WT and KO BMMSCs were treated with the NF-κB-specific inhibitor QNZ (10 nM) for another 2 h after 24-h treatment of rmCcl2 (100 ng/mL) and INCB3344 (10 nM). o The mRNA level of Lcn2 . p Lcn2 levels in the culture medium were determined by ELISA. q Illustration of how the AP-1 inhibitor modulates Ccl2 gene expression; created with BioRender ( https://BioRender.com ). r–u WT and KO BMMSCs were treated with the AP-1-specific inhibitor T-5224 (10 μM) for 24 h. r Sonicated BMMSCs were subjected to ChIP assay using an anti-c-Jun antibody. Representative gel images of end-point ChIP-qPCR products are shown. s Quantitative results are shown as the percentage of input DNA. t The mRNA level of Ccl2 . u Ccl2 levels in the culture medium were determined by ELISA. v Luciferase reporter assay was performed in isolated WT and KO BMMSCs transfected with pGL3 constructs containing the mouse wild-type Ccl2 promoter (Ccl2-WT) or the mutated Ccl2 promoter with mutations in the c-Jun binding motif (Ccl2-Mut, sequence in supplementary Fig. ). The firefly luciferase activity was measured and normalized to Renilla luciferase activity. n = 5 for each group. The data are presented as the means ± SEMs; * p < 0.05, ** p < 0.01, *** p < 0.001. See also supplementary Figs. –

Article Snippet: To investigate the regulatory effects of AP-1 inhibitor on Ccl2, 10 μM AP-1 inhibitor T-5224 (MCE, #HY-12270) was supplemented into the medium of WT or Piezo1 KO BMMSCs for 24 h. To investigate the regulatory role of NF-κB on Lcn2 expression, WT and PDGFRα-Piezo1 KO BMMSCs were first treated with recombinant mouse Ccl2 protein (rmCcl2, 100 ng/mL) and CCR2 antagonist INCB3344 (10 nM) for 24 h, followed by supplementation with the NF-κB-specific inhibitor QNZ (10 nM, MCE, #HY-13812) for additional 2 h. To investigate the effects of Klf2 on BMMSC differentiation, 40 MOI of lentivirus encoding eGFP or eGFP together with Klf2 (WZ Biosciences Inc) was used to infect WT or Piezo1 KO BMMSCs for 72 h with the presence of 5 μg/mL polybrene before differentiation.

Techniques: Expressing, Inhibition, Activation Assay, Isolation, Gene Expression, Real-time Polymerase Chain Reaction, Recombinant, Sonication, Chromatin Immunoprecipitation, Amplification, ChIP-qPCR, Luciferase, Reporter Assay, Transfection, Construct, Binding Assay, Sequencing, Activity Assay, Translocation Assay, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Software