Journal: The American journal of pathology

Article Title: CXCL1-Chemokine (C-X-C Motif) Receptor 2 Signaling Stimulates the Recruitment of Bone Marrow-Derived Mesenchymal Cells into Diffuse-Type Gastric Cancer Stroma.

doi: 10.1016/j.ajpath.2016.07.024

Figure Lengend Snippet: Figure 2 The effect of conditioned medium (CM) from gastric cancer cells on the migration activity of BM-MCs. A: Representative image of migrating BM-MCs into an 8-mm-pore membrane filter. B: CM from DGC cell lines, OCUM-2MD3, OCUM-12, OCUM-2MLN, KATO-III, NUGC3, and NUGC4, significantly increased the number of migrating BM-MCs, but the non-DGC cell lines MKN74 and SNU16 had no effect. Six randomly chosen fields were counted for each assay, and the mean of six fields was calculated as the sample value. C: Profiles of cytokines in conditioned medium from culture cells. Antibodies of 102 cytokines were spotted on nitrocellulose membranes. CXCL1 (1), CXCL5 (2), lipocalin-2 (3), CXCL8 (4), Dkk1 (5), CCL-20 (6), and EMMPRIN (7) were commonly detected in DGC cell lines, OCUM-2MD3, OCUM-12, OCUM-2MLN, KATO-III, and NUGC3, but not in the non-DGC cell lines MKN74 and SNU16. D: Relative ratio of positive control of above seven cytokines by semiquantitative analysis. Consistent with the qualitative result, the above seven cytokines were more expressed in DGC cell lines than in non-DGC cell lines. n Z 4 (B). ***P < 0.001 versus control. Original magnification: 200 (A). CCL, chemokine (C-C motif) ligand; DMEM, Dulbecco’s modified Eagle’s medium.

Article Snippet: A neutralizing antibody for CXCL1, anti-human CXCL1 antibody, was purchased from R&D Systems.

Techniques: Migration, Activity Assay, Membrane, Positive Control, Control

Journal: The American journal of pathology

Article Title: CXCL1-Chemokine (C-X-C Motif) Receptor 2 Signaling Stimulates the Recruitment of Bone Marrow-Derived Mesenchymal Cells into Diffuse-Type Gastric Cancer Stroma.

doi: 10.1016/j.ajpath.2016.07.024

Figure Lengend Snippet: Figure 3 Determination of BM-MC migration-stimulating cytokines from cancer cells. A: Chemotaxis assay of BM-MCs with seven cytokines spe- cifically detected in DGC cell lines. B: Wound-healing assay with cultured mediums from DGC cell lines and above seven cytokines. The mean of eight fields was calculated as the sample value. C: Effect of anti-CXCL1 antibody on the migration-stimulating activity of DGC cells in BM-MCs. D and E: Transfection of CXCR2 single guide RNA (sgRNA) by CRISPR/Cas9-mediated genome editing successfully decreased the expression of chemokine (C-X-C motif) receptor (CXCR) 2 mRNA and protein of BM-MCs. F: Effect of CXCR2 sgRNA transfection for BM-MCs on the migration-stimulating activity of scirrhous-type cancer cells. G: CXCL1 production in four DGC cell lines and two non-DGC cell lines. More than 1 ng/mL of CXCL1 was expressed in all DGC cell lines, not in non-DGC cell lines. n Z 4 (A, C, and F); n Z 3 (B, D, and G). *P < 0.05, **P < 0.01, and ***P < 0.001 versus control; yP < 0.05, yyP < 0.01 versus without CXCL1 antibody; zP < 0.05, zzP < 0.01 versus with negative sgRNA. CCL, chemokine (C-C motif) ligand; DMEM, Dulbecco’s modified Eagle’s medium.

Article Snippet: A neutralizing antibody for CXCL1, anti-human CXCL1 antibody, was purchased from R&D Systems.

Techniques: Migration, Chemotaxis Assay, Wound Healing Assay, Cell Culture, Activity Assay, Transfection, CRISPR, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Interstitial-resident memory CD8 + T cells sustain frontline epithelial memory in the lung

doi: 10.1084/jem.20190557

Figure Lengend Snippet: Distinct chemokine receptor usage by T RM and T EM cells upon steady-state migration to the lung airways. (A) Expression of chemokine receptors on NP-specific CD8 + T cells in the lung interstitium of mice on day 30 after x31 infection (i.n.). (B) Frequencies of chemokine receptor–expressing cells and intensities of chemokine receptor expression on indicated populations of host and partner NP-specific CD8 + T cells in the lung interstitium at day 14 after surgery. (C) CXCL10- and CXCL16-specific chemotaxis of host and partner NP-specific CD8 + T cells isolated from the lung interstitium. (D) Immunofluorescence of CXCL16 in lung tissues of mice on day 30 after x31 infection (i.n.). Pan-cytokeratin, green; CXCL6 or isotype control, red; CD45, blue. Scale bars, 100 µm. (E) Expression of indicated mRNAs isolated from each cell fraction in the lung at indicated time points (see Fig. S1). (F) Experimental design for G and H. Congenic mice were infected i.n. with x31 and subjected to parabiotic surgery 30 d later. Pairs of mice were treated i.n. with either anti-CXCL16 neutralization Ab or CXCR3 blockade Ab from day 37 after infection. Abs were administered every other day for 1 wk. (G) Ratios of CD11a + cells among host and partner NP-specific CD8 + T cells in the lung airways after Ab treatment. (H) Host/partner ratios of NP-specific CD8 + T cells in the lung airways after Ab treatment. Data are representative of two independent experiments (mean and SD of four [B–D and F–H] to five [A and E] mice per group). Error bars represent mean ± SD of four (B–D and F–H) or five (A and E) mice per group. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test (B and C), and unpaired t test (G and H). MFI, mean fluorescent intensity; PI, post-infection.

Article Snippet: An optimal concentration of either extracellular domain of recombinant CXCL16 (500 μg/ml; R&D Systems) or recombinant murine CXCL10 (10 −8 M; Peprotech) was prepared in the serum-free medium (Hybridoma-SFM Complete DPM; Gibco) and added to the lower chamber.

Techniques: Migration, Expressing, Infection, Chemotaxis Assay, Isolation, Immunofluorescence, Control, Neutralization

Journal: The Journal of Experimental Medicine

Article Title: Interstitial-resident memory CD8 + T cells sustain frontline epithelial memory in the lung

doi: 10.1084/jem.20190557

Figure Lengend Snippet: Schematic model of the maintenance of CD8 + T RM and T EM cells in the lung. In the lung interstitium, CD8 + T RM cells (a major population) are predominantly localized in the RAMD, while CD8 + T EM cells (a minor population) distribute sparsely in unaffected areas. Reactivation of CD8 + T RM cells leads to up-regulation of CXCR6, which drives continual migration to the airway in response to CXCL16 expressed constitutively in the lung airways. Some CD8 + T RM cells in the RAMD undergo homeostatic proliferation to compensate for the continuous loss of cells by intraepithelial migration. Lung interstitium CD8 + T RM cells are primarily maintained independently of CD8 + T EM cells, although a low level of T EM to T RM cell conversion may occur. While tissue-circulating CD8 + T EM cells in the lung interstitium leave tissues via lymphatics, some cells migrate to the airways in response to CXCR3 ligands. Airway memory CD8 + T cells are continuously replenished by new cells recruited from the lung interstitium. Red text indicates new findings from our current study.

Article Snippet: An optimal concentration of either extracellular domain of recombinant CXCL16 (500 μg/ml; R&D Systems) or recombinant murine CXCL10 (10 −8 M; Peprotech) was prepared in the serum-free medium (Hybridoma-SFM Complete DPM; Gibco) and added to the lower chamber.

Techniques: Migration

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 2 SR-PSOX/CXCL16 plays a role in phagocytosis of bacterial components and the production of interleukin 12 (IL-12) by macrophages. Thioglycollate-elicited peritoneal macrophages from SR-PSOX/CXCL16 knockout (KO) and wild-type (WT) mice were subjected to an ex vivo phagocytosis assay against bacteria. (A) Microscopic observation of phagocytosis was performed in peritoneal macrophages from WT mice (upper column) and SR-PSOX/CXCL16 KO mice (lower column). The upper and lower panels of each column show light microscopic findings and fluorescent images, respectively. Scale bars, 25 mm. (B) Fluorescence intensity, from which the fluorescence values of the no-cell background control wells were subtracted, was measured using a microplate reader at the indicated times. The statistical comparison of fluorescence intensity between WT (open circles) and SR-PSOX/CXCL16 KO macrophages (filled circles) was assessed by repeated measure analysis of variance followed by unpaired Student t test. (C) Peritoneal macrophages from WT (open bars) and SR-PSOX/CXCL16 KO mice (filled bars) were incubated with 500 U/ml interferon g (IFNg) for 16 h, followed by stimulation with 100 ng/ml lipopolysaccharide (LPS) or 30 mg caecal bacterial lysate (CBL) for 24 h, and the culture supernatants were analysed by ELISA to measure the concentrations of IL-6 and IL-12/23 p40. The results are expressed as means6SEM of the data from three independent experiments. The statistical difference was determined by unpaired Student t test. *p<0.05 and **p<0.01 between SR-PSOX/ CXCL16 KO and WT macrophages.

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Knock-Out, Ex Vivo, Phagocytosis Assay, Bacteria, Fluorescence, Control, Comparison, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 3 SR-PSOX/CXCL16 levels are higher in mice with dextran sulfate sodium (DSS)-induced colitis. (A) The serum levels of SR-PSOX/ CXCL16 in mice with DSS-induced colitis on day 8 and control mice were measured by ELISA. (B). The gene expression of SR-PSOX/CXCL16 in colonic tissues with or without DSS-induced colitis was determined by quantitative real-time reverse transcriptionePCR (RTePCR) and was normalised to glyceraldehyde phosphate dehydrogenase (GAPDH). (C). The production of SR-PSOX/CXCL16 in colonic tissues with or without DSS-induced colitis was investigated by western blot analysis. (D) SR- PSOX/CXCL16 concentrations in supernatants of colon fragment cultures were measured by ELISA. The results are expressed as means6SEM (n¼10 in each group). (A), (B) and (D) The statistical difference was determined by unpaired Student t test. *p<0.05 and **p<0.01 between mice with DSS-induced colitis and normal controls.

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Control, Enzyme-linked Immunosorbent Assay, Gene Expression, Western Blot

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 4 SR-PSOX/CXCL16 is expressed predominantly on macrophages in colonic tissues of mice with dextran sulfate sodium (DSS)-induced colitis. (A) Immunostaining was performed in Peyer’s patches as a positive control, normal colons and colons with 3% DSS-induced colitis. Serial sections of each tissue were stained with H&E, control goat immunoglobulin G (IgG) and anti-mouse SR-PSOX/CXCL16 monoclonal antibody. (B). Immunofluorescent staining was performed in normal colons and colons with 3% DSS-induced colitis using antibodies against CD11b (green), SR-PSOX/CXCL16 (red) and control goat IgG. The merged images and their magnified images are shown. Scale bars, 50 mm (A), 20 mm (B, left 3 lanes) and 10 mm (B, right lane).

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Immunostaining, Positive Control, Staining, Control

Journal: Gut

Article Title: SR-PSOX/CXCL16 plays a critical role in the progression of colonic inflammation.

doi: 10.1136/gut.2010.221879

Figure Lengend Snippet: Figure 5 Activity of dextran sulfate sodium (DSS)-induced colitis is lower in SR-PSOX/CXCL16 knockout (KO) mice. (A) Serial change in body weight in SR-PSOX/CXCL16 KO and wild-type (WT) mice with or without 3% DSS-induced colitis. Data are expressed as the percentage change from the starting body weight. (B) Representative image and colonic length in SR-PSOX/CXCL16 KO and WT mice with or without 3% DSS-induced colitis on day 8. (C) Representative histological findings and the scores of colonic inflammation of SR-PSOX/CXCL16 KO and WT mice withor without 3% DSS-induced colitis on day 8. Scale bars, 100 mm. (D) Colonic tissues of SR-PSOX/CXCL16 KO and WT mice with or without 3% DSS-induced colitis were incubated with fluorescein isothiocyanate (FITC)-conjugated CD3, CD11b and CD11c, followed by nuclear counterstaining with 4‘,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 mm. (E) Fluorescent in situ hybridization analysis was performed with colonic tissues of SR-PSOX/CXCL16 KO and WT mice with or without 3% DSS-induced colitis using eubacterial oligonucleotide probe EUB-338 (red), followed by DAPI (blue). Scale bars, 50 mm (left two lanes) and 20 mm (right lane). (F) Colonic macrophages from SR-PSOX/CXCL16 KO and WT mice were subjected to an ex vivo phagocytosis assay against bacteria. Scale bars, 25 mm. (G) MLN cells from SR-PSOX/CXCL16 KO and WT mice on days 0, 5 and 8 after administration of 3% DSS were cultured with immobilised anti-CD3 plus CD28. Supernatants were collected after 72 h and subjected to ELISA to measure the concentration of interferon g (IFNg) and interleukin 17 (IL-17). (A)e(D), (F) and (G) The results are expressed as means6SEM (n¼10e12 in each group). The statistical comparison was assessed by repeated measure analysis of variance followed by unpaired Student t test (A) and (F). The statistical difference was determined by unpaired Student t test (B), (D) and (G) or ManneWhitney U test (C). *p<0.05 and **p<0.01 between SR-PSOX/CXCL16 KO mice and WT mice with DSS-induced colitis.

Article Snippet: The sections were incubated with fluorescein isothiocyanate (FITC)-conjugated anti-CD11b mAb (1:200; BD Pharmingen) and biotinylated anti-CXCL16 antibody (1:1000; R&D Systems) or goat IgG isotype control overnight at 48C.

Techniques: Activity Assay, Knock-Out, Incubation, In Situ Hybridization, Ex Vivo, Phagocytosis Assay, Bacteria, Cell Culture, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison

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

Article Title: Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy.

doi: 10.4049/jimmunol.180.4.2367

Figure Lengend Snippet: FIGURE 1. The transcription and translation of chemokine CXCL16 in human first-trimester trophoblasts. The real-time quantitative PCR was used to analyze transcription of CXCL16 in trophoblasts and JAR cells with cyclophilin A (CyP) as positive control. Three independent experiments were done (including 15 placental samples) and the results were reproducible (A). The specific brown-colored stainings for CXCL16 were recognized in the cytoplasm and cytomembrane of the primary-cultured villous cytotrophoblasts (B), and extravillous cytotrophoblasts (C) by immunocytochemistry, and the positive stainings were recognized by villous cytotrophoblasts and ST (E) and by extravillous cytotrophoblasts (F) by immunohistochemistry. No background staining was observed in goat isotype controls (D and G). The experiments were repeated five times with five placenta samples, respectively. The picture is a representative one. Magnification: B, D, E, and G, 200; C and F, 400.

Article Snippet: The human CXCL16 ELISA kit (R&D Systems) was used to measure chemokine production in each supernatant and CM according to the manufacturer’s instructions.

Techniques: Real-time Polymerase Chain Reaction, Positive Control, Cell Culture, Immunocytochemistry, Immunohistochemistry, Staining

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

Article Title: Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy.

doi: 10.4049/jimmunol.180.4.2367

Figure Lengend Snippet: FIGURE 2. The accumulated concentration of CXCL16 in culture me- dium of first-trimester cytotrophoblasts was examined by ELISA. Purified cytotrophoblasts were seeded at 1 106, 5 105, 1 105, 5 104, 1 104, and 5 103 cells/ml, and then the supernatants were collected and measured after 12, 24, 36, 48, 60, 72, and 100 h of culture. The results showed CXCL16 accumulation in the medium during the course of culture. Each point represents the mean SD obtained from duplicate dishes, and the data are representative of two independent experiments.

Article Snippet: The human CXCL16 ELISA kit (R&D Systems) was used to measure chemokine production in each supernatant and CM according to the manufacturer’s instructions.

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

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

Article Title: Human trophoblasts recruited T lymphocytes and monocytes into decidua by secretion of chemokine CXCL16 and interaction with CXCR6 in the first-trimester pregnancy.

doi: 10.4049/jimmunol.180.4.2367

Figure Lengend Snippet: FIGURE 6. Correlation of CXCL16 chemotaxis to CXCR6 expression level in the peripheral (A) and decidual (B) immune cells. CXCL16 che- motaxis was shown as the ratio of the number of migrated immune cells treated by CXCL16 (100 ng/ml) to the number of migrated immune cells in the control.

Article Snippet: The human CXCL16 ELISA kit (R&D Systems) was used to measure chemokine production in each supernatant and CM according to the manufacturer’s instructions.

Techniques: Chemotaxis Assay, Expressing, Control

Journal: Nature Communications

Article Title: Targeting cardiomyocyte ADAM10 ectodomain shedding promotes survival early after myocardial infarction

doi: 10.1038/s41467-022-35331-0

Figure Lengend Snippet: a Principal components analysis (PCA) of transcriptomes of sham-operated (Sham, n = 3), GI254023X (A10i, n = 4) and DMSO ( n = 4) treated mice 3 days after myocardial infarction. Principal components were computed for each sample using gene expression. The first two principal components (PC1 and PC2) are shown. The percent of variance explained by each component is reported in parentheses. b Volcano plot representing the expression changes of all genes. Significantly down- and upregulated genes (FDR < 0.01) are colored navy and magenta, respectively. Genes that do not show significant expression changes are colored gray. Examples of neutrophil chemotaxis-associated genes are labeled with gene names. P -values were calculated using the R packages DESeq2 R (v1.30.1) and IHW (1.18.0) . Functional annotation clustering for the gene ontology (GO) terms c biological process and d molecular function of genes with significantly decreased expression upon A10i treatment (Fisher exact test with Benjamini-Hochberg posttest). e , f Gene set enrichment analysis of genes with significantly decreased expression upon A10i treatment performed on GO terms. Gene set enrichment analysis was performed using the GSEA tool (v4.1.0) of the Broad Institute and the DESeq2 R package (v1.30.1) . g In-depth analysis of the significantly downregulated genes of the indicated functional annotation clusters. h Quantification of CXCL1 ( n = 4 vs. 5, P = 0.380952), CXCL5 ( n = 8 vs. 8, P = 0.633877), CXCL16 ( n = 10 vs. 8, P = 0.572604), CX3CL1 ( n = 9 vs. 9, P = 0.00399), IL-1β ( n = 8 vs. 8, P = 0.000155) and IL-6 ( n = 6 vs. 5, P = 0.452381), serum levels in A10i and DMSO treated mice 3 days after MI (mean ± SEM, ns not significant, ** P < 0.01, *** P < 0.001, two-tailed Mann–Whitney test). i Western blot analysis and j , k quantification of j CX3CL1 and k IL-1β in heart tissue lysates of sham-operated (Sham) and LAD-ligated (MI) mice 3 days after infarction ( n = 5, mean ± SEM, ns not significant, * P < 0.05, ** P < 0.01, Kruskal–Wallis test with Dunn’s posttest, exact P -values are provided in the Source Data file). l Western blot analysis and m quantification of CX3CL1 in heart tissue lysates from patients with ischemic cardiomyopathy (ICM) and non-failing controls (NF) ( n = 6, mean ± SEM, ** P = 0.0031, two-tailed t test). Source data are provided as a Source Data file.

Article Snippet: Monoclonal or polyclonal antibodies against ADAM8 (1:1000, 23778-1-AP, Proteintech), ADAM10 (1:1000, ab124695, Abcam), ADAM10 (1:1000, AB19026, Millipore), ADAM12 (1:1000, 14139-1-AP, Proteintech), ADAM17 (1:1000, 24620-1-AP, Proteintech), ADAM19 (1:1000, NBP1-69364, Novus biologicals), CXCL16 (1:1000, MAB503, R&D Systems), CX3CL1 (1:1000, AF472, R&D Systems), Fas-L (1:1000, sc-957, Sant Cruz), GAPDH (1:5000, sc-365062, Santa Cruz), IL-1β (1:1000, #12242, Cell Signaling), N-Cadherin (1:1000, 610921, BD Biosciences), Notch1 (1:1000, ab8925, Abcam), TNFα (1:500, #11948, Cell Signaling) were used.

Techniques: Gene Expression, Expressing, Chemotaxis Assay, Labeling, Functional Assay, Two Tailed Test, MANN-WHITNEY, Western Blot

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 1. CXCL16 and CXCR6 are expressed by astrocytes, microglia, and neurons. A, RT-PCR analysis for CXCL16 and CXCR6 mRNAinadultbraintissuesofwtandcxcr6 gfp/gfpmiceandwtprimarycellcultures as indicated (left), Western blot analysis for

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 3. CXCL16 protects hippocampal neurons from excitotoxic cell death. A, CXCL16 concentration–survival response rela- tionship. Primary hippocampal neurons were exposed to Glu (100 M, 30 min) in the presence or in the absence of CXCL16 (at indicatedconcentrations),andafter18hanalyzedforcellsurvival(seeMaterialsandMethods).ResultsrepresentthemeanSEM (n5–9).B,Exampleofimmunohistochemicalanalysisofcleavedcaspase-3(green),NeuNorGFAP(red)expressionincontrol, Glu-challenged and Glu/CXCL16-treated mixed hippocampal cultures. Upon Glu challenge there is an increase in the number of caspase-3 positive cells that are also positive for NeuN with respect to control or Glu/CXCL16-treated cultures. Nuclei are stained withHoechst(blue);whitearrowsindicatecaspase-3-positiveneurons;whitestarsindicatecaspase-3-negativeneurons;images acquiredwith40objective.C,HistogrambarofcellsurvivaluponGluinsultinwtandcxcr6 gfp/gfpmice.Incxcr6 gfp/gfphippocam- palcultures,CXCL16(100nM)isnotabletoincreaseneuronalsurvivalupontoxicinsult.ResultsrepresentthemeanSEM(n 4). Data are expressed as the percentage of viable cells in treated cultures taking as 100% the number of viable cells in wt control condition.Statisticalanalysis:one-wayANOVAfollowedbyHolm–Sidakposthoctest*p0.05(A);two-wayANOVAfollowedby Holm–Sidak post hoc test *p 0.05 (C).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Concentration Assay, Control, Staining

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 4. CXCL16 acts on astrocytes to promote neuroprotection. A, Minocycline (MC) pre- vented CX3CL1- but not CXCL16-induced neuroprotection upon Glu insult. Hippocampal cul- tures were preincubated or not with MC (200 nM, 30 min) and then stimulated for 30 min with Glu, Glu/CXCL16 (100 nM), or Glu/CX3CL1 (100 nM) in the presence or in the absence of MC, as indicated. In normal condition (nil) both CXCL16 and CX3CL1 increased neuronal survival upon Glu insult; in the presence of MC only CX3CL1 failed to protect neurons. Results represent the mean SEM (n 4–5). Data are expressed as percentage of viable cells in treated cultures takingas100%thenumberofviablecellsincontrolnilcondition.B,AstrocytesmediateCXCL16 neuroprotection.Left,Whenhippocampalcellsfromcxcr6 gfp/gfpmicewerecoculturedwithwt microgliaandstimulatedwithGlu,CXCL16wasineffectiveoncellsurvival.Resultsrepresentthe mean SEM (n 4). Data are expressed as the percentage of viable cells in treated cultures taking as 100% the number of viable cells in microglia control condition. Right, When hip- pocampal cells from cxcr6 gfp/gfp mice were cocultured with wt astrocytes, in presence or ab- sence of MC, CXCL16 determined increased neuronal survival. Results represent the mean SEM (n 3–7). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in astrocytes control condition. Statistical analysis: one-way ANOVA followed by Holm–Sidak post hoc test *p 0.05 (A, B).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Control

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure5. CXCL16neuroprotectionrequiresA3Ractivity.A,CXCL16neuroprotectionrequiresADOactivity.Toremoveextracel- lularADO,primaryrathippocampalcultureswereincubatedwithADA(1U/ml,1hbefore,during,andfollowingGluinsult).While inthepresenceofADO(niltreatment)CXCL16preventedneuronalcelldeath;inabsenceofADO(ADAtreatment)CXCL16wasnot able to promote neuronal survival. To prevent ADO activity hippocampal cultures were incubated with the generic AR antagonist CGS15943(100nM).UponthistreatmenttheprotectiveeffectofCXCL16wassignificantlyreducedcomparedwithcontrolcondition (niltreatment).ResultsrepresentthemeanSEM(n4–11).Dataareexpressedaspercentageofviablecellsintreatedcultures takingas100%thenumberofviablecellsincontrolnilcondition.B,EffectsofpharmacologicalinhibitionofARs.Rathippocampal neuronsweretreatedwithspecificantagonists(DPCPX,50nM;SCH58261,5nM;MRS1706,20nM;MRS1523,100nM)andusedfor Glu-excitotoxic experiments, as indicated. Only in the presence of MRS1523 was CXCL16 neuroprotection significantly reduced compared with the control condition (nil). Results represent the mean SEM (n 5–9). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in control nil condition. C, Genetic deletion of A3R preventedCXCL16effect.HippocampalneuronsobtainedfromwtorA3R /micewerestimulatedwithGluinthepresenceorin theabsenceofCXCL16.InA3R /cultures,CXCL16failedtoincreasecellsurvivaluponGluinsult.Resultsrepresentthemean SEM (n 4). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in wt controlcondition.Statisticalanalysis:one-wayANOVAfollowedbyHolm–Sidakposthoctest;*p0.05(A–B);two-wayANOVA followed by Holm–Sidak post hoc test *p 0.05 (C).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Activity Assay, Incubation, Control

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 6. The release of CCL2 by astrocytes upon CXCL16 stimulation is determinant for CXCL16 neuroprotection. A, HEK cells thatexpressCCR2migrateinresponsetoconditionedmedium(c.m.)derivedfromCXCL16stimulatedastrocytes.Thec.m.derived fromastrocytestreatedwithvehicle,CXCL16(100nM)or2-CL-IB-MECA(100nM),wascollectedafter18h.Thec.m.ofCXCL16-and 2-CL-IB-MECA-treatedastrocytesspecificallyincreasedthechemotaxisofHEK-expressingCCR2(CCR2-HEK).Resultsrepresentthe mean SEM (n 8–10). B, CXCL16 stimulation induces CCL2 release from astrocytes. Astrocytes were incubated with CXCL16 (100nM,30min)orvehicleandthemediawerereplacedandcollectedafter6h.CCL2levelsinthemediaweremeasuredbyELISA. ResultsrepresentthemeanSEM(n7).C,NeutralizationofCCL2activitysignificantlypreventedCXCL16neuroprotection.In primary hippocampal cultures treated with neutralizing -CCL2 Ab (3 g/ml; 30 min before, during, and after Glu challenge), neuronalsurvivalfollowingCXCL16stimulationwasreducedcomparedwithsurvivalobtainedincontrolcondition(IgGtreatment; 3 g/ml). Results represent the mean SEM (n 5–8). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in IgG control condition. D, CCL2 is able to reduce Glu-excitotoxic cell death. Primary hippocampal cultures were exposed to Glu (100 M; 30 min) in the presence or in the absence of CCL2 (100 nM) as indicated, and analyzed for cell survival. Results represent the mean SEM (n 5–6). Statistical analysis: one-way ANOVA followed by Holm–Sidak post hoc test, *p 0.05 (A, C, D); Student’s t test *p 0.05 (B).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Derivative Assay, Incubation, Control