ccl12  (R&D Systems)

Journal: Journal of Medical Entomology

Article Title: Mouse chymase mast cell protease-4 facilitates blood feeding of Aedes aegypti (Diptera: Culicidae) mosquitoes

doi: 10.1093/jme/tjaf137

Figure Lengend Snippet: Sensitized serum enhances mast cell degranulation and chymase production, and chyamse mMCP-4 degrades some Aedes aegypti salivary proteins (SGPs). A) β-hexosaminidase or B) chymase enzyme activity was measured in BMMCs incubated with serum from mosquito-sensitized or naïve mice and stimulated with SGPs at the indicated concentrations (0, 1, 10, or 100 ng/ml). Lipopolysaccharide (1 μg/ml) was used as a positive control. “+” or “−” indicates the presence or absence of non-sensitized serum, sensitized serum, and SGPs for each condition. Data are presented as means ± SEM from 2 independent experiments with 3 technical replicates each ( n = 6 per condition). C) 20 μg of the extracted SGPs were incubated with 0.01 μg of recombinant mouse tryptase MCP-6 or chymase MCP-4 for overnight (approximately 18 h). SDS-page gels were stained with coomassie blue. Proteins marked with arrow were identified by liquid chromatograph mass spectrometer. Statistical analysis was performed using ordinary 1-way ANOVA followed by Sidak’s multiple comparisons test for preselected pairs. * P < 0.05; ** P < 0.01; **** P < 0.0001; ns, not significant.

Article Snippet: To evaluate the enzymatic degradation of mosquito SGPs by mouse mast cell proteases, 20 μg of SGPs were incubated with either 0.01 μg of recombinant mouse tryptase MCP-6 (Cat# 3736-SE, R&D Systems) or 0.01 μg of recombinant mouse chymase MCP-4 Reactions were carried out at room temperature (∼22 °C) for approximately 18 h to allow for proteolytic cleavage.

Techniques: Activity Assay, Incubation, Positive Control, Recombinant, SDS Page, Staining, Mass Spectrometry

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Integrated analysis of chemokine/cytokine protein after 9-Gy myeloablative radiation, HSCs, and adoptive cellular therapy and in vitro migration assays and single-cell RNA sequencing analysis in KR158B glioma. (A) Cytokine and chemokine profiling of glioma protein lysates in the tumor secretome in adoptive cellular therapy–treated gliomas and untreated control. Statistical comparisons were made using the 1-way ANOVA test. (B) CCL12 protein levels in glioma lysates were quantified by ELISA. Statistical analysis was made using the unpaired Student t test. (C) Transcriptomic analysis of chemokine ligands and receptors using nanoString digital spatial profiling. Statistical analysis was performed using the 2-way ANOVA test. (D) In vitro trans-well migration assay of MDSCs migrating in response to glioma conditioned media from excised glioma tissue combined with neutralizing antibodies against CCL2 and/or CCL12. Statistical analysis was performed using the 1-way ANOVA test. (E) ELISA CCL12 protein quantification using immune cell–specific isolates from excised glioma tissue. Statistical analysis was performed using the 1-way ANOVA test. (F) Immune cell subset deconvolution from single-cell RNA sequencing analysis from excised glioma tissue. (G) mRNA expression of CCL12 overlayed on immune cell subset deconvolution using single-cell RNA sequencing analysis from excised glioma tissue. (H) In vitro trans-well migration assay of MDSCs migration in response to immune cell-specific isolates combined with either IgG or CCL12 neutralizing antibody. Statistical comparisons conducted using the 2-way ANOVA test. * P ≤ .05. ** P ≤ .01. *** P ≤ .001. **** P ≤ .0001; error bars: mean ± standard deviation. Abbreviations: ANOVA, analysis of variance; MDSCs, myeloid-derived suppressor cells.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: In Vitro, Migration, Single Cell, RNA Sequencing, Control, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Derivative Assay

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Association of Ccl2 and macrophage-associated gene expression with clinical outcomes in glioblastoma patients. (A) Kaplan-Meier analysis of disease-free survival based on Ccl2 expression. (B) Kaplan-Meier analysis of overall survival of Cd14 expression. (C) Kaplan-Meier analysis of disease-free survival stratified by Fcgr3a expression. (D) Deconvolution of single-cell RNA sequencing cell subsets in the glioma microenvironment of GBM patients. (E) Single-cell RNA sequencing deconvolution of Ccl2 expression among cell clusters from GBM patients. (F) Deconvoluted quantification of Ccl2 expression from cell clusters identified from GBM patient samples. Statistical comparisons made using the log-rank test with P values presented on the survival graphs along with hazard ratios and P values for hazard ratios.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: Gene Expression, Expressing, Single Cell, RNA Sequencing

Journal: Cell Death & Disease

Article Title: TREM2 facilitates gastric cancer progression and immune evasion via inhibiting TRIM21-mediated STAT1 degradation in tumor-associated macrophages

doi: 10.1038/s41419-025-08198-4

Figure Lengend Snippet: A , B KEGG and GSEA analysis between shTREM2 TAMs and Scramble TAMs. C Cytokines and chemokines-related genes regulated by TREM2 in RNA-seq data. D Cytokines and chemokines screening for the above genes depicted by venn diagram. ( E ) Volcano plots showing DEGs in the shTREM2 TAMs compared with scramble TAMs. |log2FC | > 1, p -value < 0.05. F Correlation analysis between TREM2 and CCL8 (upper panel) and PD-L1 (bottom panel) from TISIDB. G mRNA expression of CCL8 in 56-paired GC tumor tissues and adjacent tissues. H Representative images of CCL8 expression in paired tumors and adjacent tissues from 14 GC patients using western blot. I Representative images of the mIF staining of macrophage CCL8 and TREM2 in GC tumor tissues. J Percentage of CCL8 + macrophage based on TREM2 expression. K Correlation between macrophage CCL8 and TREM2 expression in GC tissues. Data are presented as mean ± SD. Statistical significance was analyzed using a Student’s t -test. *** < 0.001, **** < 0.0001.

Article Snippet: For the in vivo CCL8 recuse experiment, C57BL/6 mice with GC tumors were injected with 200 μg of mouse recombinant CCL8 (Cat. No. HY-P7239; MCE) or isotype control twice a week for two weeks.

Techniques: RNA Sequencing, Expressing, Western Blot, Staining

Journal: Cell Death & Disease

Article Title: TREM2 facilitates gastric cancer progression and immune evasion via inhibiting TRIM21-mediated STAT1 degradation in tumor-associated macrophages

doi: 10.1038/s41419-025-08198-4

Figure Lengend Snippet: A – D EdU and Colony formation assays for cell proliferation of GC cells cultured by conditioned media from shTREM2 TAMs, with or without recombinant CCL8. E – H Transwell and wound healing assays were used to test cellular invasion and migration of GC cells cultured by conditioned media from shTREM2 TAMs, with or without recombinant CCL8. I Gross appearance of subcutaneous GC tumors from indicated groups. J , K The tumor weight and tumor volume of each group at the end point. Data are presented as mean ± SD. Statistical significance was analyzed using one-way ANOVA test. ** < 0.01, *** < 0.001, **** < 0.0001.

Article Snippet: For the in vivo CCL8 recuse experiment, C57BL/6 mice with GC tumors were injected with 200 μg of mouse recombinant CCL8 (Cat. No. HY-P7239; MCE) or isotype control twice a week for two weeks.

Techniques: Cell Culture, Recombinant, Migration

Journal: Cell Death & Disease

Article Title: TREM2 facilitates gastric cancer progression and immune evasion via inhibiting TRIM21-mediated STAT1 degradation in tumor-associated macrophages

doi: 10.1038/s41419-025-08198-4

Figure Lengend Snippet: A Venn diagram presenting the overlapping potential unique TREM2-interacting proteins identified through LC-MS/MS analysis. B List of the top 10 potential unique proteins that interact with TREM2. C Silver staining of the gel. D , E Co-IP of endogenous TREM2 with STAT1 in TAMs. F Representative IF images of the colocalization of TREM2 and STAT1 protein. G GST pull-down assays were conducted to investigate the direct binding between TREM2 and STAT1. ( H ) Western blot of TREM2 and phosphorylated/non-phosphorylated STAT1, PD-L1, and CCL8 in the indicated TAMs. I The level of CCL8 was evaluated using western blot and ELISA in indicated groups. J The level of PD-L1 was evaluated using western blot and RT-qPCR in indicated groups. K Schematic of putative STAT1-binding sites in the CCL8 gene promoter region and the sequence logo and frequency matrix of STAT1. L ChIP PCR to investigate the binding of STAT1 to the CCL8 promoter sequences in indicated TAMs. M Relative luciferase activities of reporters containing full-length or fragments of the CCL8 promoter. N Relative luciferase activities of different reporters containing mutated sequences of the CCL8 promoter in the indicated TAMs. Data are presented as mean ± SD. Statistical significance was analyzed using a Student’s t -test and one-way ANOVA test. ** < 0.01, *** < 0.001, **** < 0.0001, ns, not significant.

Article Snippet: For the in vivo CCL8 recuse experiment, C57BL/6 mice with GC tumors were injected with 200 μg of mouse recombinant CCL8 (Cat. No. HY-P7239; MCE) or isotype control twice a week for two weeks.

Techniques: Liquid Chromatography with Mass Spectroscopy, Silver Staining, Co-Immunoprecipitation Assay, Binding Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Sequencing, Luciferase

Journal: Cell Death & Disease

Article Title: TREM2 facilitates gastric cancer progression and immune evasion via inhibiting TRIM21-mediated STAT1 degradation in tumor-associated macrophages

doi: 10.1038/s41419-025-08198-4

Figure Lengend Snippet: A Western blot of the protein and phosphorylation level of STAT1 in indicated TAMs. B Western blot of phosphorylated and non-phosphorylated STAT1, CCL8, and PD-L1 in the indicated TAMs. C , D Co-IP of exogenous SYK with STAT1 in HEK-293T. E Co-IP of endogenous SYK with STAT1 in TAMs. F Co-IP of exogenous phosphorylation of STAT1 in HEK-293T. G Co-IP of interaction between STAT1 and SYK in TREM2 overexpression and knockdown TAMs. H GST pull-down assays were conducted to investigate the direct binding between STAT1 and SYK with or without TREM2. I Co-IP of phosphorylation of STAT1 in TREM2 overexpression and knockdown TAMs. J Western blot of cytoplasmic and nuclear phosphorylated and non-phosphorylated STAT1 in TREM2 overexpression and knockdown TAMs. K , L IF images of the colocalization of TREM2 and p-STAT1 protein. M Schematic diagram showing TREM2 recruits STAT1 and promotes its phosphorylation through SYK, which, in turn, upregulates CCL8 transcription in TAMs. N , O Representative mIF images of fresh GC tumor sections. Data are presented as mean ± SD. Statistical significance was analyzed using a Student’s t -test. ** < 0.01, *** < 0.001.

Article Snippet: For the in vivo CCL8 recuse experiment, C57BL/6 mice with GC tumors were injected with 200 μg of mouse recombinant CCL8 (Cat. No. HY-P7239; MCE) or isotype control twice a week for two weeks.

Techniques: Western Blot, Phospho-proteomics, Co-Immunoprecipitation Assay, Over Expression, Knockdown, Binding Assay