ccr2 antagonist incb3344  (Selleck Chemicals)

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

Journal: Cancer Research

Article Title: Elevated Mast Cell Abundance Is Associated with Enrichment of CCR2 + Cytotoxic T Cells and Favorable Prognosis in Lung Adenocarcinoma

doi: 10.1158/0008-5472.CAN-22-3140

Figure Lengend Snippet: MCs correlate with the infiltration of CCR2 + CTLs. A, Left, heat map showing the Pearson correlation of the immune cell population by FCM. Number representing the Pearson correlation index. Right, the transcriptional correlation of the transcriptional levels of MC marker (TPSAB1) and T-cell markers (CD4, CD8A, and FOXP3). B, The expression of chemokines in MCs from nLung, gLUAD, and sLUAD samples. The shaded areas represent the upper quantile and lower quantile. C, The secretion of CCL2, CCL4, CCL5, and CXCL8 by MCs detected by Luminex. Ctr, unstimulated MCs; solid, stimulated MCs from solid LUAD; GGO, stimulated MCs from GGO. D, Representative flow plots showing CCR2 + CTLs gating (left) and proportion of CCR2 + CTLs among tumor-infiltrating CTLs (right). FMO, fluorescence minus one. n = 36 (Mann–Whitney). E, Scatterplot showing the Pearson correlation of the proportion of CCR2 + CTLs (divided by the total CTL number) and MCs (divided by the total CD45 + cells) by FCM analysis ( n = 10). F, Three-plex staining panel showing the spatial distribution of CCR2 + CTLs and MCs. G, Spatial analysis of the relationship between MCs and CTLs. Left, depiction of methodology for spatial analyses performed. The number of CTLs close to per MCs. H, The migration rate of CTLs (the ratio of migrated CTLs to total CTLs) in the transwell assay ( n = 6, paired t test). Blank group, medium only; TS group, tumor supernatants; Usti-MC group, nonstimulated MCs; and Sti-MC group, stimulated MCs. I, The migration rate of CTLs (the ratio of migrated CTLs to total CTLs) in the migration blocking assay ( n = 4, paired t test). CCR2 T − , CCR2 − CTLs; CCR2 T + , CCR2 + CTLs. *, P < 0.05; **, P < 0.01; ns, nonsignificant.

Article Snippet: To identify the potential role of the CCL2/CCR2 axis in mediating the migration of CTLs, sorted MCs were preincubated with or without the CCL2 antagonist (Carlumab, CNTO 888, 30 ug/mL, MCE; ref. ) or CCR2 antagonist (INCB3344, 15 nmol/L, Selleck Chemicals; ref. ) for 30 minutes after adequate stimulation as mentioned above.

Techniques: Marker, Expressing, Luminex, Fluorescence, MANN-WHITNEY, Staining, Migration, Transwell Assay, Blocking Assay

Journal: Cancer Research

Article Title: Elevated Mast Cell Abundance Is Associated with Enrichment of CCR2 + Cytotoxic T Cells and Favorable Prognosis in Lung Adenocarcinoma

doi: 10.1158/0008-5472.CAN-22-3140

Figure Lengend Snippet: Characterization of LUAD-infiltrating CCR2 + CTLs. A, Bar plot showing the expression of costimulatory molecules. The x -axis value represents the FC of the expression in costimulation molecules on CCR2 − CTLs or CCR2 + CTLs compared with bulk CTLs ( n = 4). B, Box plot showing the expression of coinhibitory molecules ( n = 4). C, Frequency of TRM + (CD103, top; CD49a, middle; CD69, bottom) CCR2 − CTLs or CCR2 + CTLs. Representative flow plots (left) and summary (right) of four independent experiments (paired t test). D, Frequency of granzyme K expressing CCR2 − CTLs or CCR2 + CTLs ( n = 4; paired t test). E, Frequency of cytotoxic molecule–secreted CCR2 − CTLs or CCR2 + CTLs ( n = 6; paired t test). F, Left, representative flow plots showing the apoptosis of cocultured GFP-A549 cell line. Right, proportion of surviving target cells ( n = 4; Wilcoxon test). G, Representative example of LUAD section stained by mIF with anti-CCR2 (red), CD8 (green), and tryptase (blue) antibodies. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: To identify the potential role of the CCL2/CCR2 axis in mediating the migration of CTLs, sorted MCs were preincubated with or without the CCL2 antagonist (Carlumab, CNTO 888, 30 ug/mL, MCE; ref. ) or CCR2 antagonist (INCB3344, 15 nmol/L, Selleck Chemicals; ref. ) for 30 minutes after adequate stimulation as mentioned above.

Techniques: Expressing, Staining

Journal: Cancer Research

Article Title: Elevated Mast Cell Abundance Is Associated with Enrichment of CCR2 + Cytotoxic T Cells and Favorable Prognosis in Lung Adenocarcinoma

doi: 10.1158/0008-5472.CAN-22-3140

Figure Lengend Snippet: MCs correlated with therapeutic response to TKI therapy but not chemotherapy. A, Clinic relevance of CCL2 + MCs (Mann–Whitney). B, Clinic relevance of CCR2 + CTLs (two-tailed unpaired t test). C, Oncogene mutation relevance of CCL2 + MCs or CCR2 + CTLs ( χ 2 test). D, The prognostic value of CCL2 + MCs–CCR2 + CTLs in surgically resected LUAD. P values were determined by the log-rank test. E, Nomogram showing independent prognostic factors for survival in surgically resected LUAD by Cox proportional hazard analyses (*, independent prognostic factors). F, The prognostic value of CCL2 + MCs in patients with LUAD treated with chemotherapy. P values were determined by the log-rank test. G, The correlation of risk of recurrence after chemotherapy and CCL2 + MCs. The P value was determined by the χ 2 test. H, MC quantification as a proportion of immune cells (Mann–Whitney; n = 15 for the TN group; n = 14 for the RD group; and n = 20 for the PD group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant.

Article Snippet: To identify the potential role of the CCL2/CCR2 axis in mediating the migration of CTLs, sorted MCs were preincubated with or without the CCL2 antagonist (Carlumab, CNTO 888, 30 ug/mL, MCE; ref. ) or CCR2 antagonist (INCB3344, 15 nmol/L, Selleck Chemicals; ref. ) for 30 minutes after adequate stimulation as mentioned above.

Techniques: Clinical Proteomics, MANN-WHITNEY, Two Tailed Test, Mutagenesis

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Exposed to High-Dose Ionizing Radiation Promote M2 Polarization of Macrophages, Which Induce Radiosensitivity in Cervical Cancer

doi: 10.3390/cancers15051620

Figure Lengend Snippet: CCL2 is critical for high-dose irradiated CAFs to promote M2 polarization. ( A ) mCAFs were exposed to 8 Gy radiation or not (control), and the supernatant of mCAFs was subjected to multiplex cytokine array analysis 24 h later. Left: a representative blot. Right: quantification of dot intensity of significantly changed cytokines. ( B ) Cervical cancer cell lines and CAFs were irradiated with the indicated doses. After 24 h, CCL2 expression was analyzed by Western blot analysis. ( C ) mCAFs and hCAFs were exposed to the indicated doses of radiation. After 24 h, the supernatant was subjected to ELISA. Data are shown as mean ± SD from three independent experiments. The data were analyzed using one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001. ( D ) BMDMs were treated with 20 ng/mL CCL2 for 24 h and then subjected to flow cytometry analysis. Data are shown as mean ± SD from three independent experiments. Differences between groups were analyzed using unpaired Student’s t -test. * p < 0.05. ( E ) M0 BMDMs treated with 10 nM CCR2 antagonist (INCB3344) were co-cultured with irradiated or non-irradiated mCAFs for 3 days. Then, the cells were subjected to flow cytometry analysis to evaluate CD206 expression. Data are shown as mean ± SD from three independent experiments. * p < 0.05. ( F ) Anti-CCL2 neutralizing antibody (20 μg/mL) was added to a co-culture system consisting of 8 Gy irradiated mCAFs and M0 BMDMs. The BMDMs were subjected to flow cytometry analysis after 3 days of co-cultivation. Data are shown as mean ± SD from three independent experiments. The data were analyzed using one-way ANOVA. * p < 0.05. ( G ) M0 BMDMs were co-cultured with mCAFs exposed to the indicated doses of radiation for 3 days and 10 nM CCR2 antagonist (INCB3344) was added into the 8 Gy irradiated mCAF co-culture system. Immunofluorescence analysis of the expression of Ym-1 (red) in BMDMs. The whole western blots are shown in File S1.

Article Snippet: The reagents used for the experiments included rm-CCL2, rh-CCL2 (PeproTech), pe-roxisome proliferator-activated receptor (PPAR)γ antagonist (GW9662; MCE), CCL2 re-ceptor (CCR2) antagonist (INCB3344; MCE), and CCL2-neutralizing antibody and isotype control IgG (Clone2H5; eBioscience, Vienna, Austria).

Techniques: Irradiation, Control, Multiplex Assay, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Cell Culture, Co-Culture Assay, Immunofluorescence

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Exposed to High-Dose Ionizing Radiation Promote M2 Polarization of Macrophages, Which Induce Radiosensitivity in Cervical Cancer

doi: 10.3390/cancers15051620

Figure Lengend Snippet: Model summarizing the proposed signaling events among CAFs, TAMs, and cervical cancer cells under high-dose radiation. CAFs secrete an elevated level of CCL2 upon treatment with high-dose radiation. CCL2 promotes pro-tumor transition in macrophages. M2 macrophages induce radioresistance in cervical cancer cells.

Article Snippet: The reagents used for the experiments included rm-CCL2, rh-CCL2 (PeproTech), pe-roxisome proliferator-activated receptor (PPAR)γ antagonist (GW9662; MCE), CCL2 re-ceptor (CCR2) antagonist (INCB3344; MCE), and CCL2-neutralizing antibody and isotype control IgG (Clone2H5; eBioscience, Vienna, Austria).

Techniques:

Journal: Anesthesiology

Article Title: Local sympathectomy promotes anti-inflammatory responses and relief of paclitaxel-induced mechanical and cold allodynia in mice

doi: 10.1097/ALN.0000000000003241

Figure Lengend Snippet: (A) Representative images and quantification of ionized calcium binding adaptor molecule 1 (IBA1) protein levels in DRG tissues of male mice with sham- and local microsympathectomy (mSYMPX) at day 7 after paclitaxel (n=6 male mice/group, t-test, p = 0.107 compared to sham). (B) The same tissues and conditions were also probed for transcriptional changes and similarly to the protein levels, the IBA1 mRNA was unchanged (n=4 male mice/group, t-test, p = 0.141 compared to sham). (C) In contrast, the macrophage markers cluster of differentiation 68 (CD68), EGF-like module-containing mucin-like hormone receptor-like 1 (EMR1), integrin alpha X (ITGAM) and C-X3-C motif chemokine receptor 1 (CX3CR1), as well as the monocyte chemotactic markers chemokine (C-C motif) ligand 2 (CCR2) and C-C chemokine receptor type 2 (CCL2) were significantly increased (n=4 male mice/group, t-test, *p<0.05, ***p < 0.001 compared to sham).

Article Snippet: We purchased the 6-hydroxydopamine (6-OHDA, cat# H4381) from MilliporeSigma (Burlington, MA), TGFβ (cat# TP723438) from Origene (Rockville, MD), the SB431542 (cat# S1067), a TGFβ inhibitor, from Selleckchem, the liposomal clodronate (cat# 283539) from Liposoma (The Netherlands, Amsterdam) and C-C chemokine receptor 2 (CCR2) antagonist INCB3344 (cat# A3494) from APExBIO (Houston, TX).

Techniques: Binding Assay

Journal: Anesthesiology

Article Title: Local sympathectomy promotes anti-inflammatory responses and relief of paclitaxel-induced mechanical and cold allodynia in mice

doi: 10.1097/ALN.0000000000003241

Figure Lengend Snippet: (A) Schematic illustration of the experiment showing the timeline of local microsympathectomy (mSYMPX), injections of paclitaxel (PAX), injections of INCB3344 (intravenously on day 5, 6, and 7) or liposomal clodronate (intraperitoneally on day 5 and 7), and behavioral and transcriptional (qPCR = real-time quantitative RT-PCR) assays. (B) Time course of paclitaxel-induced mechanical allodynia tested in ipsilateral hind paws in male mice treated with a vehicle control and INCB3344 (n=5 male mice/group, two-way ANOVA showed significant difference between mSYMPX –INCB3344 and -vehicle groups, Group × Time interaction: F4,32 = 10.14, p < 0.001; Bonferroni post hoc analysis revealed a significant difference between groups on day 6, 7 and 10. ***p < 0.001). (C) Time course of paclitaxel-induced mechanical allodynia tested in ipsilateral hind paws in male mice treated with a liposomal vehicle control and clodronate (n=5 male mice/group, two-way ANOVA showed significant difference between mSYMPX -clodronate and -vehicle groups, Group × Time interaction: F3,24 = 4.12, p = 0.017; Bonferroni post hoc analysis revealed a significant difference between groups on day 7 and 10. *p < 0.05, ***p < 0.001). (D) Liposomal clodronate treatment commonly used to deplete monocytes/macrophages showed a minimal effect on ionized calcium binding adaptor molecule 1 (IBA1) transcriptional expression in DRGs (Clodronate × Vehicle groups, p = 0.012), but a drastic transcriptional reduction of IBA1 in spleen tissues (Clodronate × Vehicle groups, p = 0.001) of mice 10 days after paclitaxel (n=4–5 male mice/group, t-test, *p<0.05, ***p < 0.001 compared to vehicle).

Article Snippet: We purchased the 6-hydroxydopamine (6-OHDA, cat# H4381) from MilliporeSigma (Burlington, MA), TGFβ (cat# TP723438) from Origene (Rockville, MD), the SB431542 (cat# S1067), a TGFβ inhibitor, from Selleckchem, the liposomal clodronate (cat# 283539) from Liposoma (The Netherlands, Amsterdam) and C-C chemokine receptor 2 (CCR2) antagonist INCB3344 (cat# A3494) from APExBIO (Houston, TX).

Techniques: Quantitative RT-PCR, Binding Assay, Expressing