Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-C. Histones (50 μg/ml, 24 hours) induced chemokine production and release as demonstrated with a Proteome Profiler™ Antibody Array in Hepa1-6 cells. D. Knockdown of NF-κB p65 and TLR4 (but not TLR2 and RAGE) in Hepa1-6 cells inhibited histone (50 μg/ml, 24 hours)-induced CCL9/10 release as demonstrated by ELISA assay (n=3, *, p<0.05 versus control shRNA group). E. Anti-CCL9/10 neutralizing antibody (1 mg/ml) partly inhibited histone (50 μg/ml, 24 hours)-induced Hepa1-6 cell migration (n=3, *, p<0.05).

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Ab Array, Knockdown, Enzyme-linked Immunosorbent Assay, Control, shRNA, Migration

Journal: Oncotarget

Article Title: Novel chemokine-like activities of histones in tumor metastasis

doi: 10.18632/oncotarget.11226

Figure Lengend Snippet: A-B. Compared with the control group, TLR4 depletion (by using TLR4 −/− mice or TLR4 knockdown cells) or inhibition of histone release (by administration of 10 mg/kg heparin or 10 mg/kg H3 neutralizing antibody) limited the formation of lung metastasis (as shown in arrow) in mice based on tail vein injection of 3×10 6 Hepa1-6 cells (N=5 mice/group, *, p<0.05 versus control group). In contrast, control IgG (10 mg/kg) did not inhibit the formation of lung metastasis (B). C. Serum nucleosome levels were reduced after treatment with heparin in wildtype, but not in TLR4 −/− mice (N=5 mice/group, *, p<0.05 versus control group). D. Conceptual relationships between histone and tumor metastasis. Histone is a nuclear DAMP and can be released during cell injury or death. Once released, histone can promote cell migration and invasion through the TLR4-ERK-NF-κB pathway, which induces chemokine production and release.

Article Snippet: The production or release of chemokines was assayed using a Proteome ProfilerTM Antibody Chemokine Array Kit (#ARY020) from R&D Systems Inc. according to the manufacturer's instructions.

Techniques: Control, Knockdown, Inhibition, Injection, Migration

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: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: CX3CR1 and HIF-1α expression by OvCa tissues: Ovarian tissues from normal and various cancer stages [well-differentiated (Stage I), moderately differentiated (Stage II), and poorly differentiated (Stage III)] were stained with anti-CX3CR1 and anti-HIF-1α antibodies. Magenta (AP) color shows CX3CR1, and Brown (DAB) color shows HIF-1α staining. An Aperio ScanScope CS system with a 40X objective captured digital images of each tissue. Representative cases are immuno-intensities of CX3CR1 and HIF-1α using image analysis Aperio ImageScope v.6.25 software

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Expressing, Staining, Software

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Expression of CX3CR1 and CX3CL1 by OvCa cells. OVCAR-3, SW 626, and TOV-112D cells were stained with FITC-conjugated or anti-CX3CR1 and PE-conjugated isotype or anti-CX3CL1 antibodies, and the expressions were quantified by flow cytometry. The gray and white histograms represent isotypes and CX3CR1/CX3CL1 fluorescence intensity, respectively

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Western blot expression of hypoxia regulatory markers in OvCa cells. Immunoblot detection of OVCAR-3, SW 626, and TOV-112D cells exposed to 3, 6, 9, or 12 h hypoxia respectively. Immunoblotting was accomplished with primary antibodies against HIF-1α, CX3CR1, EMT marker (Snail), and MMP-9. As an internal standard for equal loading, anti-GAPDH antibody was used to probe blots

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Western Blot, Expressing, Marker

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: mRNA analysis of hypoxia-induced signaling molecules in OvCa cells. Ovarian tumor cells (OVCAR-3, SW 626, and TOV-112D) were exposed to hypoxia for 3, 6, 9, or 12 h. Quantitative RT-PCR results for expressions of CX3CR1, HIF-1α, MMP9, Snail, Twist, N- and P-cadherin are shown. The data were normalized to the levels of the housekeeping gene (18 S), and the analyses were accomplished in triplicate. Data were presented as fold change in expression (± standard error); the asterisks indicate significant differences as determined by Student’s t -test (* P < 0.05; ** P < 0.01)

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Quantitative RT-PCR, Expressing

Journal: Journal of Ovarian Research

Article Title: Hypoxia-inducible factor-1α induces CX3CR1 expression and promotes the epithelial to mesenchymal transition (EMT) in ovarian cancer cells

doi: 10.1186/s13048-019-0517-1

Figure Lengend Snippet: Migration and invasion induced by the CX3CR1/CX3CL1 interaction in hypoxic OvCa cells. OVCAR-3, SW 626, and TOV-112D cells were cultured in the presence of CoCl 2 (150 μM) (to mimic hypoxia), and with or without CX3CL1 (chemoattractant) and KC7F2 (inhibitor) in the medium. Over 6 days, migration and invasion were assessed. ( a ) Morphology and ( b ) quantitative analysis of surface area, representing the invading cells projecting out of the spheroid into the medium when using CX3CL1. For cells under hypoxia, there was minimal growth, or they remained as aggregates when treated with or without the inhibitor, KC7F2

Article Snippet: After washing with PBS-T, tissue sections were incubated with streptavidin-horseradish peroxidase (HRP, Biolegend) (HIF-1α) or streptavidin-alkaline phosphatase (Jackson ImmunoResearch) (CX3CR1) and developed in diaminobenzidine and/or alkaline phosphatase red chromogen coloring agent.

Techniques: Migration, Cell Culture

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 7 The number of T cells migrating across the transwell and the protein expression levels of chemokines mediating T-cell recruitment. (A) The activat ed CD3+ T cells were seeded into transwells with simple media and conditioned media from T24 cell cultures, NK cell cultures, and NK and T24 cell cocul tures. Summary data of the cumulative numbers of CD3+ T cells that migrated across the transwell in each condition are shown (n = 3 per group). ns, not significant; *p < 0.05 ; (B) Protein expression levels of chemokines in the supernatant from T24 culture, NK culture, and NK + T24 coculture measured using ELISA (n = 3 per group). Data expressed as mean ± SD were plotted. Abbreviations: CCL: C-C-motif ligand; CXCL:C-X-C-motif ligand; XCL1:X-C-motif ligand

Article Snippet: For chemokine release assay in transwell experiment, concentrations of human chemokines in the supernatant including C-C-motif ligand 1 (CCL1), CCL2, CCL4, CCL20, C-XC-motif ligand 1 (CXCL1), CXCL2, CXCL3, CXCL8, CXCL16, and C-motif ligand 1 (XCL1) were measured using human ELISA kits from CUSABIO for CCL1 (#CSB-EL004774HU), CCL2 (#CSB-Eq.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: High and selective cytotoxicity of ex vivo expanded allogeneic human natural killer cells from peripheral blood against bladder cancer: implications for natural killer cell instillation after transurethral resection of bladder tumor.

doi: 10.1186/s13046-024-02955-7

Figure Lengend Snippet: Fig. 8 Receptors of the chemokines expressed on CD3+ T cells and T cell count migrating across the transwell after blocking CCL1/2/20 in the superna tant of coculture medium. (A) The expressions of the chemokine receptors on CD3+CD56− T cells were detected using flow cytometry (n = 3 for all mol ecules). The representative images are shown. (B) Summary data of the cumulative numbers of T cells determined using a fluorescence cell analyzer after being harvested from the lower chambers, which were added into blocking antibodies against CCL1 (0.25 µg/mL), CCL2 (40 ng/mL), or CCL20 (10 µg/ mL) (n = 3 for each). Data are shown as mean ± SD. Statistical significance was determined using an unpaired t-test. *p < 0.05. Abbreviations: CXCR: C-X-C chemokine receptor; CCR:C-C chemokine receptor; XCR:X-C chemokine receptor

Article Snippet: For chemokine release assay in transwell experiment, concentrations of human chemokines in the supernatant including C-C-motif ligand 1 (CCL1), CCL2, CCL4, CCL20, C-XC-motif ligand 1 (CXCL1), CXCL2, CXCL3, CXCL8, CXCL16, and C-motif ligand 1 (XCL1) were measured using human ELISA kits from CUSABIO for CCL1 (#CSB-EL004774HU), CCL2 (#CSB-Eq.

Techniques: Cell Counting, Blocking Assay, Flow Cytometry, Fluorescence

Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A, B: the expression of HOTAIR and CCL22 mRNA were detected by RT-qPCR. C: CCL22 protein analyzed by ELISA assay. ****, P<0.001.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: LncRNA HOTAIR promotes proliferation, invasion and migration in NSCLC cells via the CCL22 signaling pathway

doi: 10.1371/journal.pone.0263997

Figure Lengend Snippet: A: The HOTAIR and CCL22 mRNA expression examined by RT-qPCR B: Cell apoptosis detected by Flow cytometry. C: The cell proliferation tested by MTS assay. D: The CCL22 protein expression tested by ELISA. E: The cell migration and invasion detected by Transwell assay. **, P<0.05; ***, P<0.01; ****, P<0.001;ns, no significance.

Article Snippet: The concentration of CCL22 in the supernatant was tested according to the instructions of ELISA kit operation (CUSABIO, CSB-E04660h).

Techniques: Expressing, Quantitative RT-PCR, Flow Cytometry, MTS Assay, Enzyme-linked Immunosorbent Assay, Migration, Transwell Assay