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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 1. RAW cells chemotax toward CX3CL1 (fractalkine). A, Live cells were incubated with rabbit anti-CX3CR1 Ab or control IgG at 4°C before fixation and incubation with anti-rabbit IgG Ab conjugated to Alexa Fluor 488. Immunofluores- cence images are shown as well as the corresponding phase contrast im- ages (see insets). Scale bar 10 m. B, Cell migration in response to in- creasing doses of CX3CL1 was mea- sured as described in Materials and Methods. C, To distinguish between chemotaxis and chemokinesis, cell migration was assessed in response to 50 ng/ml CX3CL1 added in either the bottom chamber or the top chamber of the transmigration apparatus. D, The specificity of CX3CL1-induced chemotaxis was verified by preincu- bating the cells with 10 g/ml CX3CR1 neutralizing Ab or control IgG for 1 h before subjecting the cells to the transmigration assay. E, Cells were preincubated for 5 h with or without 250 ng/ml pertussis toxin (PTX) before measuring cell migra- tion as described above. n 3. , p 0.05 compared with the correspond- ing controls.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Incubation, Control, Migration, Chemotaxis Assay, Transmigration Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 2. CX3CL1 treatment induces a rearrangement of the actin cytoskeleton. A, RAW cells were either untreated (Untr) or treated with 50 ng/ml CX3CL1 for 1 min before fixation and staining of F-actin using phalloidin conjugated to Alexa Fluor 568. Images are representatives of at least five independent experiments. Scale bar 10 m. B, Cells were treated for various times with CX3CL1, then fixed, and total F-actin, nor- malized to the cell number, was quantitatively measured as described in Materials and Methods. n 3–7 independent determinations for each time point.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Staining

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 3. CX3CL1 stimulates tyrosine phosphorylation. A, RAW cells were treated for various times with 50 ng/ml CX3CL1, and total cell lysates were subjected to Western blotting using a phosphotyrosine-spe- cific Ab (PY) or using anti--actin Ab as a verification of equal protein loading. A Western blot representative of three independent experiments is shown. B, RAW cells were either untreated or treated with CX3CL1 for 1 min before fixation and costaining of F-actin and phosphotyrosine-contain- ing proteins. C, Murine bone marrow-derived macrophages, prepared as described in Materials and Methods, were stimulated with CX3CL1 for 1 min before costaining as shown for RAW cells. Unless otherwise noted, all images shown are z-sections collected 2.5 m above the cell-substratum interface (cell midsections) using a confocal microscope. Images noted top represent the same cells as those shown below, but were taken 5 m above the coverslip level to focus on dorsal ruffles. All images are representative of at least three independent experiments. Scale bars 10 m.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Phospho-proteomics, Western Blot, Derivative Assay, Microscopy

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 4. Syk is activated upon CX3CL1 treatment, and piceatannol inhibits CX3CL1-induced cell migration. A, RAW cells were either un- treated () or treated () with 50 ng/ml CX3CL1 for 1 min before lysis and immunoprecipitation of Syk (IP Syk) or phosphotyrosine-containing proteins (IP PY) using specific Abs. Samples were then subjected to West- ern blotting (WB) using the indicated Abs. The phosphospecific Ab used (P-Syk) specifically recognizes activated Syk (phosphorylated on residues Y519/520). Input represents the amount of Syk present in total cell lysates for each condition. Western blots representative of three independent ex- periments are shown. B, Cells were pretreated with 50 m piceatannol (Syk inhibitor) or DMSO (vehicle) and then subjected to a transmigration assay in response to CX3CL1 as described in Materials and Methods. n 3. , p 0.05 compared with CX3CL1-induced stimulation in vehicle- treated cells.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Migration, Lysis, Immunoprecipitation, Western Blot, Transmigration Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 6. Cells with reduced Syk expression show impaired migration toward CX3CL1. A, Syk shRNA- treated cell migration in response to CX3CL1 was de- termined using a Transwell assay as described previ- ously and compared with NI and scr cells. n 4. , p 0.05 compared with CX3CL1-induced migration in NI cells. B, The ability of Syk shRNA and scr cells to mi- grate in response to 20 ng/ml CSF-1 was also evaluated. n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, Migration, shRNA, Transwell Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 7. CX3CL1 activation of ERK1/2 is not required for RAW cell chemotaxis. A, RAW cells were stimulated with CX3CL1 for the indicated times before Western blotting of the corresponding Triton-soluble whole cell lysates. The activation statuses of ERK1/2 (p42/44MAPK), p38MAPK, and JNK1 were assessed using phosphospecific Abs, and the corresponding total (phosphorylation-independent) levels of MAPK expression are shown below as proof of equal protein loading. B, CX3CL1-induced ERK1/2 activation levels were compared between scrambled and Syk shRNA-treated cells. Results shown were obtained with a clone exhibiting 80% Syk protein reduction. C, RAW cells were preincubated for 1 h with 30 M PD98059 (MEK inhibitor) or with DMSO (vehicle) before being subjected to a transmigration assay. PD98059 efficacy was confirmed through its ability to abolish ERK1/2 phosphorylation (see inset). For all experiments, n 3.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Activation Assay, Chemotaxis Assay, Western Blot, Phospho-proteomics, Expressing, shRNA, Transmigration Assay

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

Article Title: Syk is required for monocyte/macrophage chemotaxis to CX3CL1 (Fractalkine).

doi: 10.4049/jimmunol.175.6.3737

Figure Lengend Snippet: FIGURE 8. CX3CL1-induced cytoskeletal reorganization is disrupted in cells with reduced Syk expression. A, The ability of cells to increase their F-actin content after a 1-min CX3CL1 stimulation was compared among NI, scr, and Syk shRNA-treated (sh) cells, as previously described. n 4 independent experiments using heterogeneous cell populations. , p 0.05 compared with NI cells. B, Scrambled and Syk shRNA-treated cells were either untreated or treated with CX3CL1 for 1 min, and their ability to exhibit F-actin-rich cell protrusions (ruffles) was compared using F-actin staining. Representative images of three independent experiments are shown. Scale bar 10 m. The extent of CX3CL1-induced ruffles in individual cells was scored as described previously (18); protrusion in- dexes were calculated as the average of at least 50 cells in three different experiments and expressed as a percentage of scr cells. n 3. , p 0.05 compared with scrambled.

Article Snippet: Recombinant mouse CX3CL1 (aa 25–105) and mouse CSF-1 were purchased from R&D Systems.

Techniques: Expressing, shRNA, Staining

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Antihuman Fractalkine Reagents Used in this Study

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Control

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: Distinguishing between cleaved and membrane-tethered fractalkine, generation of specific reagents. A: Samples of Western lysates from WT CHO-K1 and CHO-K1 cells transfected with a human fractalkine expression vector 1 along with samples of supernatant taken from fractalkine-transfected CHO-K1 cells, were run on 7.5% acrylamide gels under standard reducing conditions. Samples were transferred to nitrocellulose membranes and identical membranes probed using goat anti-fractalkine polyclonal reagent (goat α-Fkn, R&D Systems) (lanes 1–3) or chicken anti-C-peptide polyclonal reagent (chicken α-C-pep, lanes 4–6). The goat α-Fkn reagent is reactive against the chemokine domain of the molecule and specifically detects twobands at the predicted size of 95 kd (lane 2, asterisk). These two bands are also detected by the chicken α-C-pep reagent (lane 5, asterisk). In addition, these reagents discriminate between cleaved and intact forms of the molecule as the goat α-Fkn detects the cleaved form of fractalkine within transfected cell supernatant (lane 3, 85 to 90 kd), whereas the chicken α-C-pep does not (lane 6). Furthermore, the goat α-Fkn detects one larger (lane 2, 100 kd) and two smaller bands (lane 2, 75 and 66 kd) within transfected CHO-K1 samples that are not detected by the chicken α-C-pep (lane 5). The larger band may be nonspecific because it has no counterpart detected by the chicken α-C-pep. The two smaller bands may indicate partially degraded forms of fractalkine, still containing the N-terminus chemokine domain. B–I: The specificity of a range of anti-fractalkine antibodies was evaluated by immunohistochemistry. Cytospins were prepared from NIH/3T3 cells transiently transfected as above, with fractalkine (3T3-Fkn) and were stained as follows. B: 3T3-Fkn stained with mouse IgG1 control as a control for C. C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb. D: 3T3-Fkn stained with no primary antibody as a control for E and F. E: 3T3-Fkn stained with goat α-Fkn. F: 3T3-Fkn stained with chicken α-C-pep. G: 3T3-Fkn stained with rabbit IgG as a control for H and I. H: 3T3-Fkn stained with rabbit α-C-peptide. I: 3T3-Fkn stained with rabbit α-N-pep polyclonal reagent. 1 Note that although there is light nonspecific staining of the nucleus within the control sections (B, D, and F) this is in marked contrast to the strong cell surface staining in sections stained with the specific reagents. Similar results were obtained using transfected CHO-K1 cells and via immunofluorescence. Original magnification, ×400.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Membrane, Western Blot, Transfection, Expressing, Plasmid Preparation, Immunohistochemistry, Staining, Control, Immunofluorescence

Journal:

Article Title: The Transmembrane Form of the CX3CL1 Chemokine Fractalkine Is Expressed Predominantly by Epithelial Cells in Vivo

doi:

Figure Lengend Snippet: The transmembrane form of fractalkine is expressed by the human colorectal adenocarcinoma cell line, DLD-1. A: DLD-1, cells were grown to confluence on glass coverslips and stained using indirect immunofluorescence for transmembrane-expressed fractalkine using the anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; green) mAb and rabbit anti-C-peptide reagent (α-C-pep; red). Strong double labeling (orange) occurred on a subset of cells where the intracellular epitope was most strongly expressed. Lower levels of anti-chemokine domain staining could be detected on most cells. B: Anti-chemokine domain reagent specificity was demonstrated by double labeling using an isotype control antibody for the anti-chemokine mAb (green) and α-C-pep (red). α-C-pep staining was also competed out by addition of 10× molar excess of the immunizing peptide (data not shown). C: The α-Fkn (green) but not α-C-pep staining (red) couldbe competed totally by pre-incubation with a 10× molar excess of recombinant human fractalkine chemokine domain (rhFkn; 362-CX-025; R&D Systems). D: Cells were double-labeled with α-cytokeratin (clone AE1/AE3, DAKO; green). Original magnifications, ×400 (A–D). E: Total RNA was prepared from DLD-1 and HUVECs cultured with or without 10 U/ml TNF-α. RNA was reverse-transcribed and triplicate 25 ng cDNA samples subjected to PCR reactions using primers specific for fractalkine (Fkn) or HPRT. There was no fractalkine or HPRT signal amplified in reverse transcriptase samples (data not shown). F: DLD-1 cells were permeabilized and stained using i) mouse α-Fkn (clone 51636.11) mAb or control mouse IgG1 mAb (Serotech), ii) goat α-Fkn polyclonal or 10% goat serum, iii) α-C-pep or rabbit IgG, iv) α-N-pep polyclonal 1 or rabbit IgG, and fractalkine expression analyzed by FACS. The bold trace shows the fluorescence of cells stained with the specific antibody, whereas the normal trace shows the background fluorescence of cells stained with the control reagent.

Article Snippet: B: 3T3-Fkn stained with mouse IgG 1 control as a control for C . C: 3T3-Fkn stained with mouse anti-fractalkine chemokine domain (mouse α-Fkn, clone 51636.11; R&D Systems) mAb.

Techniques: Staining, Immunofluorescence, Labeling, Control, Incubation, Recombinant, Cell Culture, Reverse Transcription, Amplification, Expressing, Fluorescence

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: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells shape microglia effector functions through the release of CX3CL1.

doi: 10.1002/stem.1174

Figure Lengend Snippet: Figure 4. Silencing of CX3CL1 in MSC modulates the expression of effector genes in microglia upon activation. MSC were transfected with small interfering RNA for CX3CL1 for 24 hours and utilized in the culture with N9 in the presence of LPS as described in Materials and Meth- ods. The gene expression of TNF, CX3CR1, NURR1, IL1b, EP2, and IGF1 was measured by real time polymerase chain reaction on LPS-acti- vated N9 in the presence of MSC (light gray bars) or in the presence of MSC silenced for CX3CL1 (dark gray bars). White bars represent microglia in resting condition and black bars microglia activated with LPS. Results are shown as mean 6 SD of three independent experiments. *, p < .05; **, p < .01 by t test. Abbreviations: CX3CR1, fractalkine receptor; EP2, prostaglandin E2 receptor; IL1b, interleukin1b; IGF1, insulin growth factor 1; LPS, lipopolysaccharide; MSC, mesenchymal stem cell; NURR1, nuclear receptor 4 family; TNF, tumor necrosis factor.

Article Snippet: Quantitative analysis of CX3CL1 was performed by ELISA utilizing the Quantikine Mouse CX3CL1/fractalkine Immunoassay (R&D Systems) on supernatants derived from MSC cultured for 24 hours in the presence of either 5 ng/ml IFNc or 1 lg/ml LPS and in the presence of both stimuli according to the manufacturer’s instructions.

Techniques: Expressing, Activation Assay, Transfection, Small Interfering RNA, Gene Expression, Real-time Polymerase Chain Reaction

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells shape microglia effector functions through the release of CX3CL1.

doi: 10.1002/stem.1174

Figure Lengend Snippet: Figure 5. CX3CL1 secreted by MSC can induce functional changes on activated microglia. MSC were cultured with the same concentration of N9 cells (8 105 cells) in the presence of 1 lg/ml of LPS and with (dark gray bars) or without (gray bars) 10 lg/ml of a blocking monoclonal anti-CX3CL1 antibody for 24 hours. Ca2þ i, phagocytic activity and TREM2 mRNA expression were measured as described in Materials and Methods. Blocking of release of CX3CL1 by MSC (dark gray bar) significantly inhibited the increase of intracellular Ca2þ concentration observed when microglia was activated with LPS in the presence of MSC (light gray bar) as determined by fluorimetric quantification (left upper panel). Similarly, CX3CL1 blockade on MSC in coculture with N9 (dark gray bar) reverted the enhanced phagocytic activity (middle panel and lower panels) and the upregulation in the expression of TREM2, quantified by real time polymerase chain reaction (Right upper panel), of LPS- activated microglia observed in the presence of MSC (light gray bar). Control LPS-activated N9 cells are depicted as black bars while resting microglia is shown as white bar. Results are shown as mean 6 SD of at least three independent experiments. *, p < .01; **, p < .05 by t test. The images were representative of results obtained in three independent experiments. Abbreviations: aCX3CL1, anti-CX3CL1 antibody; LPS, li- popolysaccharide; MSC, mesenchymal stem cell; TREM2, triggering receptor expressed on myeloid cells-2.

Article Snippet: Quantitative analysis of CX3CL1 was performed by ELISA utilizing the Quantikine Mouse CX3CL1/fractalkine Immunoassay (R&D Systems) on supernatants derived from MSC cultured for 24 hours in the presence of either 5 ng/ml IFNc or 1 lg/ml LPS and in the presence of both stimuli according to the manufacturer’s instructions.

Techniques: Functional Assay, Cell Culture, Concentration Assay, Blocking Assay, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Control

Journal: Stem cells (Dayton, Ohio)

Article Title: Mesenchymal stem cells shape microglia effector functions through the release of CX3CL1.

doi: 10.1002/stem.1174

Figure Lengend Snippet: Figure 6. Exogenous CX3CL1 mimics the effect of mesenchymal stem cell on activated microglia. 5 ng/ml of recombinant CX3CL1 was added to LPS-activated N9 cells (gray bars), and the expression of TNF, IL1b, CX3CR1, NURR1, EP2, and TREM2 in microglia was analyzed by real time polymerase chain reaction while Ca2þ i and phagocytic activity were measured as described in Materials and Methods and compared to LPS-activated N9 (black bars) and resting microglia (white bars). Results are shown as mean 6 SD of at least three independent experiments. *, p < .05; **, p < .05 by t test. Abbreviations: CX3CR1, fractalkine receptor; EP2, prostaglandin E2 receptor; IL1b, interleukin1b; LPS, lipo- polysaccharide; NURR1, nuclear receptor 4 family; TNF, tumor necrosis factor; TREM2, triggering receptor expressed on myeloid cells-2.

Article Snippet: Quantitative analysis of CX3CL1 was performed by ELISA utilizing the Quantikine Mouse CX3CL1/fractalkine Immunoassay (R&D Systems) on supernatants derived from MSC cultured for 24 hours in the presence of either 5 ng/ml IFNc or 1 lg/ml LPS and in the presence of both stimuli according to the manufacturer’s instructions.

Techniques: Recombinant, Expressing, Real-time Polymerase Chain Reaction, Activity 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: Journal of Immunology Research

Article Title: Diet-Induced Obesity Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma via CX3CL1/CX3CR1 Axis

doi: 10.1155/2022/5665964

Figure Lengend Snippet: DIO increased expression level of CX3CL1 and CX3CR1 in liver metastasis of PDAC. (a) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 10 6 Panc02 cells ( n = 6/group). (b) Representative immunohistochemical images of CX3CL1 staining in liver of ND and DIO. Scale bar, 20 μ m. (c) Bar graph showed the relative CX3CL1 staining positive rate. (d) Relative mRNA levels of CX3CL1 in liver of ND mice and DIO mice after intrasplenic injection with 1 × 10 6 Panc02 cells ( n = 6/group). (e) IHC staining images of CX3CR1. Scale bar, 20 μ m. (f) Bar graph showed the relative CX3CR1 staining positive rate. ∗ P < 0.05, ∗∗ P < 0.01, and ∗∗∗ P < 0.001.

Article Snippet: The membrane was blocked with 5% ( m / v ) skim milk and incubated overnight at 4°C with primary antibodies: CX3CR1(1 : 1000, Proteintech,13885-1-AP), CX3CL1 (1 : 1000, Proteintech, 10108-2-AP), and GAPDH (1 : 10000, Abways, AB2000).

Techniques: Expressing, Injection, Immunohistochemical staining, Staining, Immunohistochemistry

Journal: Journal of Immunology Research

Article Title: Diet-Induced Obesity Promotes Liver Metastasis of Pancreatic Ductal Adenocarcinoma via CX3CL1/CX3CR1 Axis

doi: 10.1155/2022/5665964

Figure Lengend Snippet: CX3CL1 promoted recruitment of CX3CR1-positive pancreatic tumor cells. (a) mRNA levels of CX3CR1 in PDAC cells. (b) Western blot analysis of CX3CR1 in human PDAC cell lines. (c) Flow cytometry analysis of the surface expression level of CX3CR1 in different human cell lines. (d)The expression level of soluble CX3CL1 was examined by using ELISA. Protein samples were obtained from the mouse liver. (e) Effect of CX3CL1 on the migratory ability of mouse PDAC cell lines Panc02; relative transferred cell numbers were analyzed. Cell migration experiments in blocker group were performed by using the blocking antibody of CX3CL1. The neutralization dose (ND 50 ) is 1 μ g/ml in the presence of 30 ng/ml mouse recombinant CX3CL1. Scale bar 10 μ m. (f) Effect of CX3CL1 on the migratory ability of human PDAC cell lines AsPC-1 and Capan-1. Different concentration of CX3CL1 recombinant protein was used in cell transwell experiments; relative transferred cell numbers were analyzed. The neutralization dose (ND 50 ) is 3 μ g/ml in the presence of 100 ng/ml human recombinant CX3CL1. Scale bar 10 μ m. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001.

Article Snippet: The membrane was blocked with 5% ( m / v ) skim milk and incubated overnight at 4°C with primary antibodies: CX3CR1(1 : 1000, Proteintech,13885-1-AP), CX3CL1 (1 : 1000, Proteintech, 10108-2-AP), and GAPDH (1 : 10000, Abways, AB2000).

Techniques: Western Blot, Flow Cytometry, Expressing, Enzyme-linked Immunosorbent Assay, Migration, Blocking Assay, Neutralization, Recombinant, Concentration Assay

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