Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 1 RT-PCR analysis of CXCL10, CXCL9, and CXCL11 gene expression in freshly isolated Langerhans cells (fLC), 48 h cultured LC (cLC), interferon-c (IFN-c)-stimulated cLC (c-cLC), freshly isolated splenic dendritic cells (fDC), 48 h cultured splenic DC (cDC), and IFN-c- stimulated cDC (c-cDC). LC and splenic DC were purified and cul- tured for 48 h in the absence or presence of 100 ng per mL of IFN-g. Samples from fresh and cultured LC and splenic DC were collected and mRNA expression was analyzed using specific primers for each chemokine. CXCL10, CXCL9, and CXCL11 mRNA expression was hardly detectable in fLC. In cLC, mRNA expression of CXCL10 and CXCL11, but not CXCL9, was induced. CXCL9 mRNA was strongly expressed only in g-cLC. In addition, mRNA for CXCL10 and CXCL11 was also strongly expressed in g-cLC. In splenic DC, mRNA for these T helper 1(Th1)-type chemokines was almost undetectable both in fDC and cDC. When stimulated with IFN-g, CXCL10, CXCL9, and CXCL11 mRNA expression was induced. Data are representative of three inde- pendent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Reverse Transcription Polymerase Chain Reaction, Gene Expression, Isolation, Cell Culture, Expressing

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 2 Production of T helper 1-type chemokines during culture of Langerhans cells (LC) and splenic dendritic cells (DC). Purified LC and splenic DC were cultured with or without interferon-g (IFN-g), and the concentration of CXCL10, CXCL9, and CXCL11 was measured at different time points (0, 12, 24, 36, 48 h) in the supernatants by ELISA. Representative data of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 3 Chemotaxis of mCXCR3-transfected 2B4 T cells. Langerhans cells (LC) and splenic dendritic cells (DC) were cultured for 48 h with 100 ng per mL of interferon-g (IFN-g) and the supernatants were collected. The culture supernatants were preincubated with or without neutralizing anti-chemokine monoclonal antibody (mAb) indicated in the figure for 30 min, and assessed for chemotactic activity to CXCR3 transfectant. RPMI 10 medium alone served as a negative control. RPMI 10 medium containing recombinant chemokine served as a positive control. The supernatants of IFN-g-stimulated LC and splenic DC exhibited chemo- tactic activity to CXCR3 transfectant, which is mediated at least by CXCL10 and CXCL9. Mean (SD) (n ¼ 3). Data are representative of three independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Chemotaxis Assay, Transfection, Cell Culture, Activity Assay, Negative Control, Recombinant, Positive Control

Journal: Journal of Investigative Dermatology

Article Title: Differential Production of Th1- and Th2-Type Chemokines by Mouse Langerhans Cells and Splenic Dendritic Cells

doi: 10.1111/j.0022-202x.2004.23607.x

Figure Lengend Snippet: Figure 7 Regulation of T helper 1-type chemokines produced by La- ngerhans cells (LC) and splenic dendritic cells (DC). LC (&) and splenic DC (’) were purified and cultured (1.5 106 cells per mL per 200 mL in each well) for 48 h with or without various stimuli. The supe- rnatants were collected and the concentration of CXCL10 (a), CXCL9 (b), and CXCL11 (c) was measured by ELISA. CXCL10 production by LC was induced by interferon-g (IFN-g), interleukin (IL)-12, lipopoly- saccharide (LPS), Staphylococcus aureus Cowen 1 (SAC), and poly- inosinic–polycytidylic acid (Poly(I:C)). In the case of CXCL9 and CXCL11, only IFN-g induced their production by LC. In the case of splenic DC, the production of CXCL10, CXCL9, and CXCL11 was in- duced by IFN-g, IL-18, LPS, and Poly(I:C). Results are the mean (SD) (n ¼ 4). Significant increase (po0.05) compared with the unstimulated group. Data are representative of four independent experiments.

Article Snippet: Measurement of CXCL9, CXCL11, and CCL17 Culture supernatants were collected, stored at 201C, and subjected to the quantification of protein levels of CXCL9, CXCL11, and CCL17 by ELISA using commercially available mouse CXCL9, CXCL11, and CCL17 immunoassay kits (R&D Systems), respectively, according to the manufacturer’s instructions.

Techniques: Produced, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: CD11c+ Tbet+ B cells constrain obesity- and vaccination-induced germinal center B cells and T helper cells

doi: 10.1101/2025.09.01.673552

Figure Lengend Snippet: Representative flow cytometry contour plots of PD-1 x CXCR5 expression within CD25-CD4+ T cells isolated from NCD (left) and HFD (right) mouse spleen (top) and liver (bottom) ( A ). Blue gates represent PD-1 HI CXCR5+ cells, yellow gates represent PD-1 HI CXCR5-cells, and black gates represent PD-1-cells ( A ). Frequency and absolute number of PD-1 HI CXCR5+ T FH cells ( B , blue), or PD-1 HI CXCR5-T PH cells ( C , yellow) within the CD25-CD4+ T cell compartments of spleen (top) and liver (bottom) of NCD (grey) and HFD (blue/yellow) mice. Quantification of Bcl-6 ( D ), ICOS ( E ), and T-bet ( F ) protein expression (gMFI) in splenic (left) or hepatic (right) CD25-CD4+ T cell subsets. Representative flow cytometry contour plots depicting relative expression of IL-21 and IFNg by PD-1 HI CXCR5+ T FH cells or PD-1 HI CXCR5-T PH cells from spleen (top) or liver (bottom) that are also CXCR3+ (blue/red) or CXCR3-(black) ( G ). Summary of frequency of IL-21 ( H,J; left) or IFNg ( I,K ; right) producing CD4+ PD-1 HI CXCR5+ T FH (blue) or PD-1 HI CXCR5-T PH (red) cells categorized by CXCR3+ expression as noted in spleen ( H, I ) or liver ( J,K ) from HFD-fed mice. Data pooled from 2 independent experiments with 4 female mice/group, Bar ± SEM; [ Student’s t-test (two-tailed) (B,C); one-way ANOVA (D-F; H-K)]; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Article Snippet: C57BL/6J WT female mice were injected IP with 0.25 mg of anti-mouse CXCR3 (CD183) and anti-mouse CXCL9 (MIG) or hamster IgG (BioXCell) every other day for 14 days after completing 12 weeks on HFD.

Techniques: Flow Cytometry, Expressing, Isolation, Two Tailed Test

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 mediates the regulation of chemokine biosynthesis in M1-polarized macrophages, as determined by RNA-seq. ( A ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h and subjected to RNA sequencing analysis. The KEGG database was used to identify enriched signaling pathways whose activation significantly differed (up). Single-gene GSEA was conducted using the GSE57065 , GSE65682 , and GSE95233 datasets to identify the relationship between PD-L1 and chemokine signaling pathways (down). ( B ) Intersection analysis of upregulated genes between the PD-L1 HI /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + LPS + IFN-γ group and between the PD-L1 NC /THP-1-M + LPS + IFN-γ group and PD-L1 NC /THP-1-M + PBS group via a Venn diagram, followed by further intersection with chemokine datasets, revealed three highly expressed PD-L1-mediated chemokines (CCL8, CXCL9, and CXCL10) under LPS + IFN-γ stimulation. ( C ) RNA sequencing analysis revealed the expression of CCL8, CXCL9 and CXCL10 upon PD-L1 overexpression. ( D ) PD-L1 NC /THP-1-M and PD-L1 HI /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( E ) Human monocytes were induced into macrophages, transfected with scramble siRNA and PD-L1 siRNA, and then treated with LPS + IFN-γ for 12 h. CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). ( F ) PD-L1 NC /THP-1-M and PD-L1 KO /THP-1-M cells were treated with LPS + IFN-γ for 12 h, after which CCL8 and CXCL9 mRNA and protein levels were quantified ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for D, E and F was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: RNA Sequencing, Protein-Protein interactions, Activation Assay, Expressing, Over Expression, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: TLR4 acts as a critical upstream regulator of PD-L1 in modulating CCL8 and CXCL9 expression. ( A ) GSEA of RNA-seq data to investigate the relationship between PD-L1 expression and Toll-like receptor signaling pathway activation. ( B ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TLR4 siRNA, respectively, followed by 12 h of stimulation with LPS (100 ng/mL) + IFN-γ (20 ng/mL). The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for B was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Expressing, RNA Sequencing, Activation Assay, Transfection

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 binds to the TLR4 downstream molecule TRAF6 to regulate CCL8 and CXCL9 expression. ( A ) Venn diagram analysis of proteins coimmunoprecipitated with PD-L1 in THP-1-M cells stimulated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 6 h via IP‒MS. After excluding proteins bound to the IgG control group, 289 PD-L1-specific interacting proteins were identified (up). Representative MS/MS spectrum showing peptide fragments of the TRAF6 protein identified in the immunoprecipitation complex (down). ( B ) THP-1-M (up) and human monocyte-derived (down) macrophages were treated with or without LPS+IFN-γ for 6 h and then harvested for Co-IP. TRAF6 protein levels were measured via Western blotting. ( C ) PD-L1 HI /THP-1-M and PD-L1 NC /THP-1-M cells were transfected with scramble siRNA or TRAF6 siRNA, respectively, followed by 12 hours of stimulation with LPS+IFN-γ. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). ( D ) Structural models of full-length PD-L1 and TRAF6 predicted by AlphaFold (PAE plots were obtained from AlphaFold). ( E ) Molecular docking generic matching information. ( F ) A protein‒protein interaction cartoon model generated by PyMOL (left). Predicted binding sites between PD-L1 and TRAF6 (right). ( G ) PD-L1 NC /THP-1-M, PD-L1 HI /THP-1-M and PD-L1 Δ62-139 /THP-1-M cells were treated with LPS+IFN-γ for 12 h. The supernatants and cells were harvested for concurrent measurements of CCL8 and CXCL9 protein and mRNA levels via ELISAs and qRT‒PCR ( n =3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C and G was performed by one-way ANOVA with LSD test for post hoc analyses. * p <0.05.

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Expressing, Control, Tandem Mass Spectroscopy, Immunoprecipitation, Derivative Assay, Co-Immunoprecipitation Assay, Western Blot, Transfection, Generated, Binding Assay

Journal: Inflammation

Article Title: Programmed death-ligand 1 (PD-L1) Modulates Chemokine Production Via the TLR4/TRAF6 Signaling Axis During LPS + IFN-γ-Induced Endotoxemia-mimicked Sepsis

doi: 10.1007/s10753-025-02394-2

Figure Lengend Snippet: PD-L1 knockout attenuates septic mouse mortality and suppresses CCL8 and CXCL9 expression. ( A – C ) PD-L1 WT /mice and PD-L1 KO /mice were intraperitoneally injected with LPS (15 mg/kg) and IFN-γ (10 µg/kg) to induce endotoxemia-induced sepsis. ( B ) The survival rate was determined over a 60-hour observation period and is presented as a Kaplan‒Meier survival curve ( n = 15 mice per group). The results are presented as Kaplan–Meier survival curves. The results of the statistical analysis were analyzed with the log-rank test. * p < 0.05. ( C ) Peripheral blood was collected 12 h posttreatment for serum isolation and PBMC separation. CCL8 and CXCL9 protein levels in serum and their corresponding mRNA levels in PBMCs were subsequently analyzed ( n = 3 independent biological replicates). ( D ) Peritoneal macrophages (PMs) were extracted from PD-L1 wild-type and knockout mice and then treated with LPS (100 ng/mL) + IFN-γ (20 ng/mL) for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). (E) Peripheral blood monocytes were extracted from PD-L1 wild-type and knockout mice and then treated with LPS + IFN-γ for 12 h. The cells were harvested for CCL8 and CXCL9 mRNA and protein detection by qRT‒PCR and ELISAs ( n = 3 independent biological replicates). The data are presented as the mean ± SEM. Statistical analysis for C was performed by Student’s t test, and that for D and E was performed by one-way ANOVA with LSD test for post hoc analyses. * p < 0.05

Article Snippet: CCL8 and CXCL9 concentrations in cell supernatants or plasma were quantified using ELISA kits (Boster Biological Technology; Wuhan, China) with absorbance measurements at 450 nm, following the manufacturer’s protocols.

Techniques: Knock-Out, Expressing, Injection, Isolation

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: Decomposition of the inflammatory score was conducted by estimating the most variable jacobians (first order partial derivative of inflammatory age) ( A ). Both positive and negative contributors to inflammatory age are observed. The top 15 most variable jacobians are CXCL9, EOTAXIN, Mip-1α, LEPTIN, IL-1β, IL-5, IFN-α and IL-4 (positive contributors) and TRAIL, IFN-β, CXCL1, IL-2, TGF-α, PAI-1 and LIF (negative contributors) (A). In a validation study, 97 healthy adults (age 25-90) well matched for cardiovascular risk factors, were selected form a total of 151 recruited subjects. Immune protein analysis was conducted in samples from these subjects. CXCL9, HGF, CXCL1 and LIF were found to change in the same direction in both the Stanford 1KIP and the validation cohort ( A and B ). Cardiovascular age was estimated using 3 parameters: (1) aortic pulse wave velocity, a measure of vascular stiffness; (2) relative wall thickness (RWT), a measure of ventricular remodeling, and (3) early diastolic mitral annular velocities (e’), a measure of ventricular relaxation. After adjusting for age, sex, BMI, heart rate, systolic blood pressure, fasting glucose and total cholesterol to HDL ratio, positive correlations were obtained between CXCL9 and PWV ( R = 0.22) and RWT ( R = 0.3) ( P < 0.05), and negative correlations were observed between LIF and PWV ( R = −0.27), and RWT ( R = −0.22) ( C and D ). No variable included in the models had high co-linearity as suggested by variance inflation factors (VIF) < 3 for each factor. Induced pluripotent stem cells (hiPSCs) were obtained from isolated fibroblasts ( N = 5, in duplicates) using the Yamanaka factors and differentiated them into endothelial cells (hiPSC-ECs) under well-defined conditions as previously described . Expression levels of CXCL9 and SIRT3 were measured by RT-PCR as described under Methods. A significant age-dependent increase in CXCL9 mRNA expression levels is observed ( P < 0.01), which reaches a plateau after the sixth cell passage ( E ). Concomitant with the increase in CXCL9, down-regulation in SIRT3 mRNA can be observed after the second cell passage ( P < 0.01) (E). Addition of increasing doses (10 to 800 ng/ml) of exogenous CXCL9 to young (day 7) hiPSC-ECs induces down-regulation of SIRT3 mRNA expression (F). Expression of the CXCL9 receptor, CXCR3, was measured in young cardiomyocytes derived from hiPSCs (hiPSC-CM) as well as in hiPSC-ECs, HUVEC cells, freshly isolated fibroblasts and hiPSCs. Elevated expression is observed in hiPSC-ECs, HUVEC cells but not in other cell types ( F ) suggesting that the endothelium but no other cell subsets is target of CXCL9 and potentially other CXCR3 ligands as well.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques: Biomarker Discovery, Isolation, Expressing, Reverse Transcription Polymerase Chain Reaction, Derivative Assay

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: To validate the association between CXCL9 expression and SIRT3 in hiPSC-ECs, CXCL9 was knockdown (KD) in hiPSCs using shRNA. Quantitative PCR data show significant knockdown of CXCL9 in hiPSCs following transfection with CXCL9-shRNA compared to scramble controls ( A ). hiPSC-ECs failed to show an increase in CXCL9 expression in CXCL9-KD cells as they were passaged when compared to scramble controls ( B ). On the contrary, CXCL9-KD hiPSC-ECs show reversal of their SIRT3 levels in serially passaged hiPSC-ECs when compared to scramble controls ( C ). Quantification of the number of capillary-like networks formed by scramble- and CXCL9-KD hiPSC-ECs show that CXCL9-KD hiPSC-ECs retain their capacity to form tubular networks even at later passages when compared to scramble controls that showed impaired tube formation at later passages of hiPSC-ECs ( D ). Line graph of percent relaxation of mouse thoracic aortic sections to Acetylcholine show impaired vascular reactivity to increasing concentrations of CXCL9, suggesting CXCL9 impairs vascular function ( E ). Significance of impaired vascular reactivity was determined by 2-way ANOVA, followed by a Bonferroni post-test, N=3, n=3-4, *p<0.05.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques: Expressing, Knockdown, shRNA, Real-time Polymerase Chain Reaction, Transfection

Journal: bioRxiv

Article Title: An Inflammatory Clock Predicts Multi-morbidity, Immunosenescence and Cardiovascular Aging in Humans

doi: 10.1101/840363

Figure Lengend Snippet: Representative images of capillary-like networks from scramble- and CXCL9-KD hiPSC-ECs show that CXCL9-KD hiPSC-ECs retain their capacity to form tubes even at later passages when compared to scramble that showed impaired tube formation towards later passages of hiPSC ECs.

Article Snippet: Following normalization, the vessels were incubated with either PBS or different concentrations of recombinant mouse CXCL9 protein (R&D systems, catalog number 492-MM) for 3-4 hrs.

Techniques:

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma

doi: 10.1186/s13046-022-02564-2

Figure Lengend Snippet: Cyclin G2 in macrophages regulates CTL chemotaxis and vascular endothelial cell tube formation via CXCL9. A CXCL9 levels in the supernatants of BMDMs from WT and Ccng2 −/− C57BL/6 mice treated with IFN-γ were determined by ELISA (representing 3 independent experiments). B , C CXCL9 levels in the supernatants of THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2 and Vector and Flag-cyclin G2) treated with IFN-γ were determined by ELISA (representing 3 independent experiments). D , E CTL chemotaxis analyzed by treating conditioned medium from BMDMs isolated from WT and Ccng2 −/− C57BL/6 mice treated with or without recombinant CXCL9. Scale bar = 200 μm (representing 3 independent experiments). F Tube formation experiments showed the tube formation ability of SVEC4–10 cells treated with conditioned medium from BMDMs isolated from Ccng2 −/− C57BL/6 mice. The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 500 μm (representing 3 independent experiments). G Tube formation experiments showed the tube formation ability of HUVECs treated with conditioned medium from a THP-1 stable cell line (shcyclin G2#1). The recombinant CXCL9 was added or not added to the conditioned medium. Scale bar = 200 μm (representing 3 independent experiments). (A–C, E–G) Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SD ** p < 0.01; *** p < 0.001; ns , not significant

Article Snippet: Cell culture supernatants were collected, and human and mouse CXCL9 levels in the supernatants were quantified using the human and mouse CXCL9 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (E-EL-H6062 and E-EL-M0020c, Elabscience), respectively.

Techniques: Chemotaxis Assay, Enzyme-linked Immunosorbent Assay, Stable Transfection, Plasmid Preparation, Isolation, Recombinant

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma

doi: 10.1186/s13046-022-02564-2

Figure Lengend Snippet: Cyclin G2 influences the activation of the STAT1-CXCL9 signaling pathway. A , B CXCL9 mRNA levels in THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2 and Vector and Flag-cyclin G2) as determined by RT–qPCR. β-actin was used as an internal control (representing 2 independent experiments). C Detection of CXCL9 mRNA levels in BMDMs isolated from WT and Ccng2 −/− C57BL/6 mice by RT-qPCR. GAPDH was used as an internal control (representing 2 independent experiments). D The STAT1 and p-STAT1 (Y701) protein levels were determined in the THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2) by western blotting. β-tubulin was used as a loading control. E The STAT1 and p-STAT1 (Y701) protein levels were determined in the THP-1 stable cell lines (Vector and Flag-cyclin G2) by western blotting. β-tubulin was used as a loading control (representing 3 independent experiments). F STAT1 protein levels in the cytoplasm and nucleus of THP-1 cells were detected by western blotting (Nonsense and shcyclin G2#1). β-tubulin was used as a cytoplasmic loading control. Lamin B1 was used as a nuclear loading control. G STAT1 immunofluorescence staining for THP-1 stable cell lines (Nonsense, shcyclin G2#1, and shcyclin G2#2), representative images are shown. Scale bar = 10 μm. ( A – C ) Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SD.**** p < 0.0001

Article Snippet: Cell culture supernatants were collected, and human and mouse CXCL9 levels in the supernatants were quantified using the human and mouse CXCL9 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (E-EL-H6062 and E-EL-M0020c, Elabscience), respectively.

Techniques: Activation Assay, Stable Transfection, Plasmid Preparation, Quantitative RT-PCR, Control, Isolation, Western Blot, Immunofluorescence, Staining

Journal: Journal of Experimental & Clinical Cancer Research : CR

Article Title: Cyclin G2 in macrophages triggers CTL-mediated antitumor immunity and antiangiogenesis via interferon-gamma

doi: 10.1186/s13046-022-02564-2

Figure Lengend Snippet: Cyclin G2 knockout in macrophages attenuates the inhibitory effects of IFN-γ on colon cancer cell growth. A–C MC38 cells were mixed with BMDMs from WT and Ccng2 −/− C57BL/6 mice at a ratio of 5:1 and injected subcutaneously into C57BL/6 mice, which were then treated with IFN-γ at specific times. Gross tumors ( A ), tumor weights ( B ), and tumor volumes ( C ) were measured at the endpoint. Data were analyzed with the unpaired Student’s t-test. Data are presented as the mean ± SEM ( n = 5). D A schematic model depicting the role of cyclin G2 in macrophages after IFN-γ treatment. Upregulated cyclin G2 after IFN-γ treatment inhibited the interaction between PP2Ac and STAT1, thereby increasing the nuclear import of STAT1 and promoting CXCL9 transcription. Increased CXCL9 secretion can promote CTL chemotaxis and inhibit vascular endothelial cell angiogenesis, ultimately inhibiting tumor progression. ** p < 0.01; **** p < 0.0001

Article Snippet: Cell culture supernatants were collected, and human and mouse CXCL9 levels in the supernatants were quantified using the human and mouse CXCL9 Enzyme-Linked Immunosorbent Assay (ELISA) Kits (E-EL-H6062 and E-EL-M0020c, Elabscience), respectively.

Techniques: Knock-Out, Injection, Chemotaxis Assay