chemoattractant media  (R&D Systems)

Journal: Nature Communications

Article Title: Human single cell RNA-sequencing reveals a targetable CD8 + exhausted T cell population that maintains mouse low-grade glioma growth

doi: 10.1038/s41467-024-54569-4

Figure Lengend Snippet: A Schematic representation of the anti-PD1 (α-PD1), anti-TIGIT (α-TIGIT), and combined (α-PD1 + TIGIT) antibody treatments. Nf1 -OPG mice were treated (200 µg/dose/ i.p., twice per week) from 12 to 16 weeks of age, and tissues were analyzed at 16 weeks. The control group was injected with anti-IgG isotype control antibodies. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. B Immunohistochemistry and quantification CD8 + T cells in the entire optic nerve (IgG, n = 10 mice; α-TIGIT, n = 10 mice; α-PD1, n = 6 mice; α-PD1 + TIGIT, n = 5 mice). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0054; α-PD1, P = 0.0003; α-PD1 + TIGIT, P = 0.0032). Scale bar, 200 µm. C Ccl2 RNA expression in the optic nerves of Nf1 -OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. D Volcano plot showing fold change and P value comparing Nf1 -OPG TAM to Nf1 +/ and WT monocytes in the optic nerves of 12-week-old mice. Upregulated genes in red, downregulated genes in blue. Differential analyses were performed using gene specific analysis (GSA). E Ccr2, Cxcr3 , and Cxcr5 expression in T cell populations from 12-week-old Nf1 -OPG mouse optic nerves, color-coded by T cell type. F Cxcl9, Ccl12 , and Cxcl13 RNA expression in the optic nerves of 12-week-old WT and Nf1 -OPG mice. Data are represented relative to the WT group ( Cxcl9 ; WT n = 3, 2 pooled optic nerves per sample; Nf1 -OPG n = 4, 2 pooled optic nerves per sample; Ccl12 ; WT n = 4, 2 pooled optic nerves per sample; Nf1 -OPG n = 4, 2 pooled optic nerves per sample; Cxcl13 ; WT n = 4, 2 pooled optic nerves per sample; Nf1 -OPG n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. To evaluate statistical differences, a two-tailed non-parametric Mann–Whitney test was performed. Exact P values are indicated. ns, not significant. G Graph showing the percentage of migrated Nf1 +/- CD8 + T cells treated with medium (Control) ( n = 5), Ccl12 ( n = 6), or Cxcl13 ( n = 5). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Dunnett’s post-test correction. Exact P values are indicated. Created in BioRender. Chatterjee, J. (2024) BioRender.com/r98n914. H Ccl12 RNA expression in the optic nerves of Nf1 -OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 5, 2 pooled optic nerves per sample; α-TIGIT n = 5, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 3, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post-test correction. Exact P values are indicated (α-TIGIT, P = 0.0012; α-PD1, P = 0.0187; α-PD1 + TIGIT, P = 0.0013). I Cxcl13 RNA expression in the optic nerves of Nf1 -OPG mice treated with IgG, α-TIGIT, α-PD1 or a combination of α-PD1 and α-TIGIT antibodies. Data are represented relative to the IgG control group (IgG, n = 4, 2 pooled optic nerves per sample; α-TIGIT n = 4, 2 pooled optic nerves per sample; α-PD1, n = 4, 2 pooled optic nerves per sample; α-PD1 + TIGIT, n = 4, 2 pooled optic nerves per sample). Data are represented as mean ± SD. A one-way ANOVA test was performed followed by a Tukey’s post test correction. Exact P values are indicated (α-TIGIT, P = 0.0003; α-PD1, P = 0.0007; α-PD1 + TIGIT, P = 0.0001).

Article Snippet: 500 μl of chemoattractant media (Ccl12 [25 ng/ml, 428-P5-025- R&D systems ] and Cxcl13 [1 µg/ml, 470-BC-025- R&D systems] ) was added to the lower chamber, and the number of T cells in the lower chambers counted 6 h later.

Techniques: Control, Injection, Immunohistochemistry, RNA Expression, Expressing, Two Tailed Test, MANN-WHITNEY

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