Journal: bioRxiv

Article Title: Structural Basis of GPCR-G Protein Pre-coupling and Activation: Insights from CCR1-Gi Complex

doi: 10.1101/2024.11.01.621549

Figure Lengend Snippet: a , Comparison of the interface between the α5 helix of Gαi and the cytoplasmic region of CCR1 in the active (green) and pre-coupled (dodger blue) states. The alignment is performed using the receptor as the reference. b-c, Agonist-induced changes in luciferase signal measured by NanoBiT between CCR1-SmBiT and Gα-LgBiT ( b ), and between Gγ-SmBiT and Gα-LgBiT ( c ). Bars and error bars indicate the mean and SEM, respectively. N = 6 ( b ) and 4 ( c ) independent experiments, performed with single replicates. d, Orthogonal views of the structures of the CCR1-Gi heterotrimer complex in the active and pre-coupled states, colored by subunit. CCR1 is shown in green (active state) and dodger blue (pre-coupled state), Gαi1 in gold (active state) and yellow (pre-coupled state), Gβ in rosy brown, Gγ in light blue, and CCL15 in brown. e-f, Luciferase complementation between various GPCRs (CCR1, CCR2, CCR4, CCR5, CXCR1, CXCR2, CX3CR1, and 5HT 1 R) fused to SmBiT and Gα-LgBiT decreases in response to agonist ( e ), whereas luciferase complementation between other GPCRs (NPY2R, FPR1, FPR2, CB1, AT 1 R, and AT 2 R) fused to SmBiT and Gα-LgBiT increases in response to agonist ( f ). N = 6-8 independent experiments, performed with single replicates. g, The model of GPCR activation in which the C-terminal residues of the Gαi α5 helix (red), while disordered in the pre-coupled state (blue), play a crucial role in the transition to the fully active state (green) and subsequent G protein dissociation

Article Snippet: Angiotensin II (HY-13948) for AT1R and AT2R, Serotonin(HY-B1473) for 5HT1eR, NPY protein(HY-P71063) for NPY2R,CX3CL1 protein(HY-P7180) for CX3CR1 are from MedChemexpress.

Techniques: Comparison, Luciferase, Activation Assay

Journal: European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery

Article Title: Characterisation of fractalkine/CX3CL1 and fractalkine receptor (CX3CR1) expression in abdominal aortic aneurysm disease.

doi: 10.1016/j.ejvs.2008.01.014

Figure Lengend Snippet: Figure 1 Localisation of fractalkine receptor (CX3CR1) in AAA. Adventitia and media stained with a control antibody (AeD) and with anti-CX3CR1 antibody (E and F) with the arrows indicating representative CX3CR1þ cells. Images taken from 2 samples of AAA tissue from a total of 28 AAA tissue samples analysed. Scale bar Z 40 mm, Magnification 16 (A and B), 40 (CeD).

Article Snippet: Human recombinant TNFa was added to cultured vSMCs (10 ng/ml) and vECs (100 ng/ml) for 12e16 h.10,17 After trypsin detachment, non-stimulated and stimulated cells were washed in PBS and incubated with goat anti-human fractalkine antibody (1:25, R&D Systems, UK) or goat Ig (1:25, Dako, UK) for 1 h. After washing cells were incubated for 30 min with FITC-conjugated swine anti-goat IgG (1:50, Caltag, USA). vECs were also stained with antibodies to human CD34 (1:50 Caltag, USA),18 for 1 h followed by incubation with Streptavidin-PE (1:100, BD Biosciences, UK) for 30 min.

Techniques: Staining, Control

Journal: European journal of vascular and endovascular surgery : the official journal of the European Society for Vascular Surgery

Article Title: Characterisation of fractalkine/CX3CL1 and fractalkine receptor (CX3CR1) expression in abdominal aortic aneurysm disease.

doi: 10.1016/j.ejvs.2008.01.014

Figure Lengend Snippet: Figure 5 Upregulation of fractalkine by vascular stromal cells in response to TNFa. Primary cultures of vascular and aor- tic smooth muscle cells (vSMC, aSMC) were incubated with 10 ng/ml TNFa for 12e16 h and vascular endothelial cells (vEC) with 500 ng/ml TNFa for 12e16 h. The values shown represent the proportion of cells expressing fractalkine following stimulation.

Article Snippet: Human recombinant TNFa was added to cultured vSMCs (10 ng/ml) and vECs (100 ng/ml) for 12e16 h.10,17 After trypsin detachment, non-stimulated and stimulated cells were washed in PBS and incubated with goat anti-human fractalkine antibody (1:25, R&D Systems, UK) or goat Ig (1:25, Dako, UK) for 1 h. After washing cells were incubated for 30 min with FITC-conjugated swine anti-goat IgG (1:50, Caltag, USA). vECs were also stained with antibodies to human CD34 (1:50 Caltag, USA),18 for 1 h followed by incubation with Streptavidin-PE (1:100, BD Biosciences, UK) for 30 min.

Techniques: Incubation, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: The chemokine CX3CL1 promotes intraperitoneal tumour growth despite enhanced T-cell recruitment in ovarian cancer

doi: 10.1016/j.neo.2025.101130

Figure Lengend Snippet: CX3CL1 is an unfavourable prognostic marker in high-grade serous ovarian cancer (HGSOC) patients. A, Representative immunohistochemistry (IHC) images of high or low CX3CL1 expression in a patient cohort of high-grade serous ovarian cancer (scale bars represent 50 µm). B, Digitally analysed and quantified CX3CL1 IHC staining intensity in omental (n = 122) and peritoneal (n = 126) metastases (mean values were normalised to TMA control tissue). C, Kaplan Meier curves showing progression free survival (left) and overall survival (right) in CX3CL1 high vs. low expressing groups of HGSOC.

Article Snippet: The primary monoclonal mouse CX3CL1 antibody (#MAB3651, R&D Systems) was applied on sections for 1 h at room temperature with a 1:100 dilution in antibody diluent (#ZUC025, Zytomed Systems).

Techniques: Marker, Immunohistochemistry, Expressing, Control

Journal: Neoplasia (New York, N.Y.)

Article Title: The chemokine CX3CL1 promotes intraperitoneal tumour growth despite enhanced T-cell recruitment in ovarian cancer

doi: 10.1016/j.neo.2025.101130

Figure Lengend Snippet: Multivariate Cox regression analysis of clinical outcome in high-grade serous ovarian cancer patients (FIGO III/IV) with respect to clinical parameters and CX3CL1 expression.

Article Snippet: The primary monoclonal mouse CX3CL1 antibody (#MAB3651, R&D Systems) was applied on sections for 1 h at room temperature with a 1:100 dilution in antibody diluent (#ZUC025, Zytomed Systems).

Techniques: Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: The chemokine CX3CL1 promotes intraperitoneal tumour growth despite enhanced T-cell recruitment in ovarian cancer

doi: 10.1016/j.neo.2025.101130

Figure Lengend Snippet: CX3CL1 expression and regulation in human and murine ovarian cancer cell lines. OV-MZ-6 cells (A, B) , OVCAR-3 cells (C) , or ID8- Trp53 −/− cells (D) were stimulated for 24 h ± TNF-α (50 ng/mL) and ± TAPI-2 (50 µM) or ± GI254 (5 µM) or the corresponding solvent controls as indicated. Bar graphs on the left show soluble CX3CL1 in cell supernatant measured after 24 h by ELISA (mean ± SEM). The plots on the right show membrane-bound CX3CL1 as determined via FACS analysis. B, Immunocytochemical staining of CX3CL1 in OV-MZ-6 cells stimulated with or without TNF-α in the presence or absence of TAPI-2, visualised by confocal laser scanning microscopy (CLSM) using an Alexa 488-conjugated secondary antibody.

Article Snippet: The primary monoclonal mouse CX3CL1 antibody (#MAB3651, R&D Systems) was applied on sections for 1 h at room temperature with a 1:100 dilution in antibody diluent (#ZUC025, Zytomed Systems).

Techniques: Expressing, Solvent, Enzyme-linked Immunosorbent Assay, Membrane, Staining, Confocal Laser Scanning Microscopy

Journal: Neoplasia (New York, N.Y.)

Article Title: The chemokine CX3CL1 promotes intraperitoneal tumour growth despite enhanced T-cell recruitment in ovarian cancer

doi: 10.1016/j.neo.2025.101130

Figure Lengend Snippet: Cx3cl1 accelerates intraperitoneal tumour growth while enhancing immune infiltration. A, Expression of murine Cx3cl1 was verified in ID8- Trp53 -/- empty vector (Ctrl) and ID8- Trp53 -/- Cx3cl1 + ( Cx3cl1 + ) cells via cell supernatant ELISA measurement of untreated or TNF-α stimulated cells (24 h, 25 ng/mL). Bars represent mean ± SEM. B, Proliferation rates of ID8- Trp53 -/- Ctrl and Cx3cl1 + cells were measured via MTT assay and normalised to the baseline value at 4 h. C , Representative pictures of ascites accumulation (left) as well as tumour load on the mesentery (upper right) and diaphragm (lower right) at the time of finalization (black arrows indicate exemplary tumour nodes). D, Kaplan–Meier curves showing ascites-free (left) and overall survival (right) of C57BL/6 mice intraperitoneally injected with 1×10 7 ID8- Trp53 -/- Ctrl or Cx3cl1 + cells (differences in median survival are indicated as Δ days). E, Relative Cx3cl1 mRNA expression in mesenteric tumour tissue of ID8- Trp53 -/- Ctrl or Cx3cl1 + tumours at the end of the experiment. F, Quantification of tumour-infiltrating immune cell subsets (CD3, CD8, granzyme B, F4/80, CD206) by immunohistochemical stainings of ID8- Trp53 -/- Ctrl or Cx3cl1 + mesenteric tumour tissues. DAB positive immune cell markers (CD3, CD8, granzyme B) were manually counted in intratumoural report images. Positive macrophage subset cells (F4/80, CD206) were digitally evaluated as percentage of all cells. G, Ki67-positive proliferation status was digitally assessed as percentage of total cells. H, 1×10 7 ID8- Trp53 −/− Ctrl or Cx3cl1 + cells were implanted intraperitoneally into athymic nude mice, and onset of ascites (left) and overall survival (right) was determined and shown as Kaplan–Meier curves (differences in median survival are indicated as Δ days). I, F4/80 macrophage marker was immunohistochemically stained in mesenteric tumour tissue from athymic nude mice, and positive cells were calculated as percentage of all cells. Horizontal lines in E-G and I indicate the mean, each dot indicates data from one individual mouse. Mouse experiments were conducted with at least 7 mice per group.

Article Snippet: The primary monoclonal mouse CX3CL1 antibody (#MAB3651, R&D Systems) was applied on sections for 1 h at room temperature with a 1:100 dilution in antibody diluent (#ZUC025, Zytomed Systems).

Techniques: Expressing, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, MTT Assay, Injection, Immunohistochemical staining, Marker, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: The chemokine CX3CL1 promotes intraperitoneal tumour growth despite enhanced T-cell recruitment in ovarian cancer

doi: 10.1016/j.neo.2025.101130

Figure Lengend Snippet: Cx3cl1 inhibits tumour growth in the subcutaneous ID8 ovarian cancer model. A, ID8 luc - Trp53 -/- Control (Ctrl) and ID8 luc - Trp53 -/- Cx3cl1 + ( Cx3cl1 + ) cells were tested for Cx3cl1 protein secretion into the cell supernatant of untreated or TNF-α (24 h, 50 ng/mL) stimulated cells by ELISA. Bars represent mean ± SEM. B, Cell proliferation rates of ID8 luc - Trp53 -/- Ctrl and Cx3cl1 + cells were compared via manual counting with a Neubauer counting chamber and normalised to the baseline value at 4 h. C-G, 1×10 7 ID8 luc - Trp53 -/- Ctrl or Cx3cl1 + cells were subcutaneously inoculated into the flanks of C57BL/6 mice (n = 6 each group). C, Graph shows caliper measurement of maximal tumour diameter (mm) once a week until the first mouse reached predefined endpoints. D, Weekly flux (photons/sec) measurement of luciferase expressing tumour cells upon intraperitoneal luciferin substrate injection. E, Representative pictures of each group indicate bioluminescence signals (day 93). The pseudocolors represent the average radiance in the unit of p/s/cm 2 /sr with a maximum radiance of 5×10 5 depicted as red color. F, Representative pictures of a subcutaneously grown tumour with a reached finalization endpoint of 1.5 cm diameter ( in vivo ). G, Kaplan–Meier plot showing survival of tumour mice (differences in median survival are indicated as Δ days). H, Cx3cl1 expression at mRNA level was measured in tumour tissue via qRT-PCR and normalised to the housekeeping control. I, Intratumoural cell proliferation was assessed via digital Ki67 marker IHC analysis, and positive cells were are given as percentage of total cells. J-K, Tumour-infiltrating CD3 + T-cells and CD8 + cytotoxic T-cells ( J ) and Foxp3 + regulatory T-cells ( K ) were determined immunohistochemically. DAB positive cells were manually counted in five report images. L , Intratumoural F4/80-positive macrophages were immunohistochemically stained and digitally analysed as percent of total cells. Horizontal lines in H-L indicate the mean, each dot indicates data from one individual mouse.

Article Snippet: The primary monoclonal mouse CX3CL1 antibody (#MAB3651, R&D Systems) was applied on sections for 1 h at room temperature with a 1:100 dilution in antibody diluent (#ZUC025, Zytomed Systems).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Luciferase, Expressing, Injection, In Vivo, Quantitative RT-PCR, Marker, Staining

Journal: Neoplasia (New York, N.Y.)

Article Title: The chemokine CX3CL1 promotes intraperitoneal tumour growth despite enhanced T-cell recruitment in ovarian cancer

doi: 10.1016/j.neo.2025.101130

Figure Lengend Snippet: PARP inhibition enhances CX3CL1 secretion from human ovarian cancer cell lines. Human ovarian cancer cell lines OV-MZ-6, CAOV-3 and OVCAR-3 were stimulated with 10 µM olaparib, 10 µM niraparib or corresponding DMSO solvent control in the absence or presence of TNF-α (10 ng/mL). After 48 h, cell supernatants were harvested, and soluble CX3CL1 concentrations were assessed via ELISA.

Article Snippet: The primary monoclonal mouse CX3CL1 antibody (#MAB3651, R&D Systems) was applied on sections for 1 h at room temperature with a 1:100 dilution in antibody diluent (#ZUC025, Zytomed Systems).

Techniques: Inhibition, Solvent, Control, Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

doi: 10.4049/jimmunol.168.2.604

Figure Lengend Snippet: FIGURE 1. RT-PCR analysis of human fractalkine and MCP-1 expres- sion in SMC from human aorta. A, Serum-starved SMC were stimulated with increasing concentrations of IFN- (F), TNF- (E), or IL-1 (u) and subsequently analyzed for the expression of mRNA encoding fractalkine. B, Serum-starved SMC were stimulated with IFN-, TNF-, IL-1, or combinations of these cytokines (each 20 ng/ml) for 16 h, and mRNA encoding fractalkine (f) or MCP-1 () was quantified. C, Induction of fractalkine (F) and MCP-1 (E) was studied over time by stimulating se- rum-starved SMC with a combination of IFN- and TNF- for 0, 2, 8, 16, and 24 h. Fractalkine (FKN) and MCP-1 mRNA levels were quantified by real-time RT-PCR and expressed as a percentage of that determined for GAPDH. Data are given as the mean and SD (n 3) and were reproduced twice in separate experiments with SMC from different donors.

Article Snippet: Recombinant extracellular domain fractalkine, chemokine domain fractalkine, human TNF- , and human IFN- were purchased from R&D Systems (Oxon, U.K.).

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

doi: 10.4049/jimmunol.168.2.604

Figure Lengend Snippet: FIGURE 2. Flow cytometric analysis of fractalkine surface expression on SMC. A, Serum-starved SMC were left unstimulated (dotted line) or were stimulated with 20 ng/ml IFN- (solid line), TNF- (gray line), or a combination of both cytokines (filled histogram) for 8 h and subsequently analyzed for expression of fractalkine on the cell surface by flow cytometry using a PE-conjugated mAb to fractalkine. Histograms shown were ob- tained in one representative of three experiments. B, Serum-starved SMC were stimulated with a combination of IFN- and TNF- for 0, 2, 8, 16, and 24 h and subsequently stained with a PE-conjugated mAb to fractalkine (F) or an isotype control (E). Results are shown as the median fluorescence intensity of labeled cells and represent the mean and SD (n 3).

Article Snippet: Recombinant extracellular domain fractalkine, chemokine domain fractalkine, human TNF- , and human IFN- were purchased from R&D Systems (Oxon, U.K.).

Techniques: Expressing, Cytometry, Staining, Control, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

doi: 10.4049/jimmunol.168.2.604

Figure Lengend Snippet: FIGURE 3. Western blot and flow cytometric analysis of fractalkine cleavage by SMC. A, Serum-starved SMC received no stimulus, IFN- or TNF- alone, or a combination of both cytokines (20 ng/ml each). After 24 h of incubation conditioned media were harvested, concentrated 10- fold, and analyzed for the presence of soluble fractalkine by Western blot- ting using an antiserum to the chemokine. B, Serum-starved SMC received no stimulus or a combination of IFN- and TNF- (20 ng/ml each) and were incubated in the presence or the absence of the protease inhibitor batimastat (20 M). After 24 h of incubation conditioned media and cells were harvested separately and analyzed for the presence of the membrane- bound and soluble fractalkine by Western blotting. Shown is one repre- sentative experiment of three. C, Cells were stimulated as described in B, fixed, and stained for surface-expressed fractalkine with a PE-conjugated mAb to fractalkine (f) or probed with an IgG1 isotype control (). The median fluorescence intensity of labeled cells was recorded by flow cy- tometry. Data are given as the mean and SD (n 3).

Article Snippet: Recombinant extracellular domain fractalkine, chemokine domain fractalkine, human TNF- , and human IFN- were purchased from R&D Systems (Oxon, U.K.).

Techniques: Western Blot, Incubation, Protease Inhibitor, Membrane, Staining, Control, Labeling

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

doi: 10.4049/jimmunol.168.2.604

Figure Lengend Snippet: FIGURE 4. Induction of intracellular calcium transients in CX3CR1- transfected HEK 293 cells by SMC-conditioned media. A, CX3CR1-trans- fected HEK 293 cells were challenged with 3-fold concentrated condi- tioned media (CM) from serum-starved SMC that were left unstimulated or were costimulated with IFN- and TNF- (20 ng/ml each, for 24 h). The role of soluble fractalkine was investigated by treating the media with a fractalkine-neutralizing mAb (50 g/ml) 30 min before the assay. As a control, cells were stimulated with recombinant full-length soluble frac- talkine (5 nM) in parallel. Data represent the calcium responses over time obtained in one representative of three experiments. B, Conditioned media were harvested from serum-starved SMC that were left unstimulated or were stimulated by a combination of IFN- and TNF- (20 ng/ml each) and incubated for 24 h in the absence or the presence of 20 M batimastat. CX3CR1-transfected HEK 293 cells were challenged with serial dilutions of the 10-fold concentrated supernatants. The cellular response was calcu- lated as the maximum change in fluorescence intensity over baseline. Re- sults are shown as the mean and SD (n 3).

Article Snippet: Recombinant extracellular domain fractalkine, chemokine domain fractalkine, human TNF- , and human IFN- were purchased from R&D Systems (Oxon, U.K.).

Techniques: Transfection, Control, Recombinant, Incubation

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

doi: 10.4049/jimmunol.168.2.604

Figure Lengend Snippet: FIGURE 5. Monocyte chemotactic activity of SMC-conditioned media. A, Media were harvested from cultures of SMC that were left unstimulated (E) or were costimulated (F) with IFN- and TNF- (both 20 ng/ml) for 24 h. Conditioned media (CM) were concentrated 10-fold and subsequently assayed in various dilutions for chemotactic activity on human monocytes. B, Conditioned media of unstimulated or IFN--/TNF--stimulated SMC were pretreated with 20 g/ml blocking mAb to MCP-1 (u) or fractalkine (f) or were left untreated () for 30 min before monocyte chemotaxis assays. The neutralizing effects of both Abs (both of IgG1 isotype) on the chemotactic activity of recombinant MCP-1 (0.3 nM) or fractalkine (3 nM), respectively, were controlled in parallel. Data are shown as the mean and SD (n 3). Values that are statistically different from each other are indicated by an asterisk (p 0.05).

Article Snippet: Recombinant extracellular domain fractalkine, chemokine domain fractalkine, human TNF- , and human IFN- were purchased from R&D Systems (Oxon, U.K.).

Techniques: Activity Assay, Blocking Assay, Chemotaxis Assay, Recombinant

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

doi: 10.4049/jimmunol.168.2.604

Figure Lengend Snippet: FIGURE 6. Adhesion of THP-1 cells to SMC. Fluorescently labeled THP-1 cells were assayed for the adhesion to a confluent monolayer of SMC. A, SMC were left unstimulated or were costimulated with IFN- and TNF- (both 20 ng/ml) for 24 h. B, Stimulation of SMC with IFN- and TNF- was performed in the presence or the absence of 20 M batimastat. As a control unstimulated SMC were incubated in parallel with or without the inhibitor. The fraction of fractalkine-dependent cell adhesion was eval- uated by exposing THP-1 cells to either 50 nM soluble fractalkine (f) or 50 nM MCP-1 (A, u) or by leaving cells unexposed () for 10 min before the adhesion assay. After 2-fold washing, the fluorescence signal from the adherent THP-1 cells was recorded as relative fluorescent units (RLU) and calculated as the mean and SD (n 4). Adhesion values that are statisti- cally different from each other are indicated by an asterisk (p 0.05).

Article Snippet: Recombinant extracellular domain fractalkine, chemokine domain fractalkine, human TNF- , and human IFN- were purchased from R&D Systems (Oxon, U.K.).

Techniques: Labeling, Control, Incubation, Cell Adhesion Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Fractalkine is expressed by smooth muscle cells in response to IFN-gamma and TNF-alpha and is modulated by metalloproteinase activity.

doi: 10.4049/jimmunol.168.2.604

Figure Lengend Snippet: FIGURE 7. Inhibition of monocyte adhesion to SMC by a neutralizing Ab to fractalkine. Fluorescently labeled monocytes were assayed for the adhesion to SMC stimulated with IFN- and TNF- (both 20 ng/ml) for 24 h. The role of SMC-expressed fractalkine was examined by exposing SMC to a fractalkine-neutralizing mAb (10 and 50 g/ml; f and u, re- spectively) or an IgG1 isotype control (o and p) or leaving cells unex- posed () 10 min before the addition of monocytes. Nonadherent cells were removed by 3-fold washing. After each wash step, the fluorescence signal from the adherent monocytes was recorded as relative fluorescent units (RLU) and calculated as the mean and SD (n 4).

Article Snippet: Recombinant extracellular domain fractalkine, chemokine domain fractalkine, human TNF- , and human IFN- were purchased from R&D Systems (Oxon, U.K.).

Techniques: Inhibition, Labeling, Control

Journal: The American Journal of Pathology

Article Title: Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

doi: 10.1016/j.ajpath.2013.02.039

Figure Lengend Snippet: Primers Used for Human RT-qPCR

Article Snippet: ELISA Cell culture supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) using a human Quantikine CX3CL1 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.

Techniques:

Journal: The American Journal of Pathology

Article Title: Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

doi: 10.1016/j.ajpath.2013.02.039

Figure Lengend Snippet: Primers Used for Mouse RT-qPCR

Article Snippet: ELISA Cell culture supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) using a human Quantikine CX3CL1 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.

Techniques:

Journal: The American Journal of Pathology

Article Title: Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

doi: 10.1016/j.ajpath.2013.02.039

Figure Lengend Snippet: Primers Used for ChIP

Article Snippet: ELISA Cell culture supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) using a human Quantikine CX3CL1 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.

Techniques: Negative Control

Journal: The American Journal of Pathology

Article Title: Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

doi: 10.1016/j.ajpath.2013.02.039

Figure Lengend Snippet: The Human Endothelial Cell Biology RT 2 Profiler PCR Array Gene List

Article Snippet: ELISA Cell culture supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) using a human Quantikine CX3CL1 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.

Techniques: Control, Clinical Proteomics, Permeability, Coagulation, Reverse Transcription

Journal: The American Journal of Pathology

Article Title: Endothelial GATA-6 Deficiency Promotes Pulmonary Arterial Hypertension

doi: 10.1016/j.ajpath.2013.02.039

Figure Lengend Snippet: Summary of Results for ChIP Analysis

Article Snippet: ELISA Cell culture supernatants were subjected to enzyme-linked immunosorbent assay (ELISA) using a human Quantikine CX3CL1 ELISA kit (R&D Systems, Minneapolis, MN) following the manufacturer's instructions.

Techniques: Binding Assay, Amplification, In Vivo

Journal: eLife

Article Title: Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation

doi: 10.7554/eLife.64909

Figure Lengend Snippet:

Article Snippet: AF488-conjugated monoclonal antibody against human CX3CL1/fractalkine chemokine domain , IC365G-100UG (Research And Diagnostic Systems, Inc (R&D Systems, Inc), Minneapolis, Minnesota, United States.

Techniques: Control, Fluorescence, Flow Cytometry, Diagnostic Assay, Luminex, Software, Multiplex Assay, Functional Assay

Journal: eLife

Article Title: Convalescent COVID-19 patients are susceptible to endothelial dysfunction due to persistent immune activation

doi: 10.7554/eLife.64909

Figure Lengend Snippet:

Article Snippet: AF488-conjugated monoclonal antibody against human CX3CL1/fractalkine chemokine domain , IC365G-100UG (Research And Diagnostic Systems, Inc (R&D Systems, Inc), Minneapolis, Minnesota, United States.

Techniques: Diagnostic Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 1. Immunohistochemical analysis of CX3CL1 expression in human colorectal cancer. (A) Representative immunoreactivity of CX3CL1 in normal colonic mucosa, precancerous lesion (adenoma), and colorectal cancer. Original magnification 320. (B) Expression scores of CX3CL1 in all tested samples (normal colonic mucosa, n = 18; adenoma, n = 18; colorectal cancer, n = 100). CX3CL1 increases in adenoma and carcinoma, but decreases in stage III tumors. Red bars indicate median value. Kruskal– Wallis and Dunn’s posttest, *p , 0.05.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Immunohistochemical staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 2. CX3CL1 expression by tumor cells does not correlate with CD3+ or CD68+ infiltrating cells at the tumor invasive front. (A) Immu- nohistochemical analysis of CD3+ or CD68+ cells in CRC samples (n = 100), at the tumor-invasive margin. (B and C) Linear regression analysis. Correlation between CX3CL1 expression and the percentage of CD3+ (B) or CD68+ (C) cells. CX3CL1 expression does not correlate with lympho- cyte or macrophage infiltration. (D) Immunohistochemistry of CX3CR1 showing that in tumor tissue only few immune-infiltrating cells express CX3CR1, whereas in healthy mucosa the majority of immune cells are CX3CR1 positive. (E) Immunofluorescence of CX3CR1 and CD3 or CD68 in human tumor tissues; arrows mark the few double-positive cells. (F) Mouse model of colitis-associated cancer in CX3CR1gfp/gfp and CX3CR1+/gfp

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Expressing, Immunohistochemistry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 3. Immunohistochemical analysis of CX3CR1 expression in human CRC and correla- tion of the CX3CL1-CX3CR1 axis with clinical outcome. (A) Representative immunoreactivity of CX3CR1 in normal colonic mucosa, precancerous lesion (adenoma), and colorectal cancer. Original magnification 320. (B) Expression scores of CX3CR1 in all tested samples (normal colonic mucosa, n = 18; adenoma, n = 18; colorectal can- cer, n = 100). CX3CR1 is low in normal tissues and precancerous lesions, and strongly increases in tu- mors. Kruskal–Wallis and Dunn’s posttest, ***p , 0.001. (C) Immunofluorescence analysis showing the coexpression of CX3CL1 (red) and CX3CR1 (green) on tumor cells. The presence of several spots of colocalization is shown in the merge panel and close-up. Original magnification 320. (D and E) Kaplan–Meier curves showing disease-free (D) and disease-specific (E) survival, in stage I–III patients according to CX3CL1-CX3CR1 axis expression. Patients with axis-negative tumors (LigLow-RecLow, black line) have significantly shorter DFS and DSS (p = 0.01 and p = 0.001, respectively), compared with axis-positive tumors (LigHigh-RecHigh, red line).

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Immunohistochemical staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 4. CX3CL1 and CX3CR1 expression is reduced in lymph node metastasis. (A) Percentage of patients with lymph node metastasis at diagnosis and with metachronous metastasis (during follow- up), according to the expression of the chemokine axis by tumor cells. Patients with axis-negative tumors (LigLow-RecLow) have higher frequency of metastases compared with axis-positive tumors (LigHigh-RecHigh). (B) Immunoreactivity of CX3CL1 and CX3CR1 in lymph node metastases and cor- responding primary tumors. Original magnification 320. (C and D) Expression scores of CX3CL1 (C) and CX3CR1 (D) in lymph node metastases (n = 36) and primary tumors (n = 36). Red bars indicate median value. Expression of both CX3CL1 and CX3CR1 in metastatic cells is significantly re- duced. Mann–Whitney, *p , 0.05, ***p , 0.001. LN, lymph node; MT, metastatic tumor; PT, primary tumors.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Expressing, Biomarker Discovery, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 6. Expression of the chemokine axis CX3CL1-CX3CR1 re- duces metastatic cell dissemination in vivo. In vivo spleen–liver mouse model of metastasis. NSG mice were spleen injected with 1:1 mixture of tumor cells expressing CX3CL1 or CX3CR1 or Mock cells (n = 10 mice each group). (A) Mice with axis-negative tumors (Mockcherry-Mockgfp, black line), had higher mortality rate compared with axis-positive tumors (CX3CL1cherry-CX3CR1gfp, red line). Log rank, *p , 0.05. (B) Represen- tative histological pictures of liver metastasis from mice receiving different combinations of CX3CL1cherry- or CX3CR1gfp-expressing tumor cells (upper panels, RKO cells; lower panels, NCI-H630). Original magnifica- tion 310. (C and D) Analysis of the liver metastatic area (mean 6 SE of three experiments with RKO, one with NCI-H630). Mice with axis-nega- tive tumors (black bars) had higher number of metastasis compared with axis-positive tumors (red bars). In (D), NCI-H630 cells constitutively produce low levels of CX3CL1, and exogenous expression of CX3CR1 reconstituted the chemokine axis, resulting in fewer metastases (green bar). One-way ANOVA with Tukey’s posttest, *p , 0.05, **p , 0.01, ***p , 0.001.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: Expressing, In Vivo, Injection

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: The Fractalkine-Receptor Axis Improves Human Colorectal Cancer Prognosis by Limiting Tumor Metastatic Dissemination.

doi: 10.4049/jimmunol.1501335

Figure Lengend Snippet: FIGURE 7. FRET analysis of CX3CL1-CX3CR1 interaction in vivo. (A) Immunofluorescence analysis of RKO (left panel) and NCI-H630 (right panel) tu- mors injected in mouse spleens. CX3CL1-CX3CR1 positive-axis tumors show several spots of colocaliza- tion (inset, white asterisk) that are absent in axis-neg- ative tumors. Original magnification 320. (B and C) Phasor plots were used to calculate GFP fluorescence lifetime variation in axis-positive or -negative tumors generated with RKO (B) or NCI-H630 (C) cells: a re- duction in GFP fluorescence lifetime is present only in axis-positive tumors. Green boxes indicate normal GFP lifetime; red boxes indicate the reduced GFP lifetime. (D and E) FRET analysis of CX3CL1-CX3CR1 inter- action. Comparison between mean lifetime of “interacting” versus “noninteracting GFP” in RKO (D) and NCI-H630 cells (E): starting from CX3CL1cherry- CX3CR1gfp–derived phasor plot, the mean lifetime of GFP, in the presence or absence of Cherry (interacting or noninteracting GFP, respectively), was compared. Interacting GFP shows a significantly lower fluo- rescence lifetime in comparison with noninteracting GFP, indicating the presence of FRET events and thus a CX3CL1-CX3CR1 interaction. Student t test, ***p , 0.001.

Article Snippet: Human recombinant CX3CL1 (rhCX3CL1) was from R&D Systems.

Techniques: In Vivo, Injection, Generated, Comparison, Derivative Assay