Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 1. IL-18 induces CXCL16 expression in rat aortic smooth muscle cells. A, ASMC express IL-18R and - mRNA. Northern blot analysis was performed using 2 g of poly(A) RNA isolated from cultured ASMC. B, dose-dependent induction of CXCL16 mRNA by IL-18. Quiescent ASMC were treated with the indicated concentrations of IL-18 for 2 h, and total RNA was isolated and analyzed for CXCL16 mRNA and 28 S rRNA (loading control). C, time course studies. Quiescent ASMC were treated with 25 ng/ml IL-18 for up to 24 h, and total RNA was analyzed for CXCL16 mRNA expression. D, IL-18 induced CXCL16 protein expression. Quiescent ASMC were treated with IL-18 as in C, and cell extracts were analyzed by Western blotting for CXCL16 protein and -actin. E, IL-18-induced CXCL16 expression was not mediated by endotoxin. Quiescent ASMC were either untreated or treated with polymyxin B vehicle (H2O) or 10 g/ml polymyxin B (left three lanes) or with 25 ng/ml IL-18, 25 ng/ml IL-18 plus vehicle, or 25 ng/ml IL-18 plus 10 g/ml polymyxin B (right three lanes) for 2 h. Total RNA was analyzed for CXCL16 expression as before. F, IL-18-induced CXCL16 expression was regulated at the transcriptional level. Quiescent ASMC were treated with 25 ng/ml IL-18 for 2 h, and the nuclear RNA was isolated and analyzed for CXCL16 transcription by the run-on assay. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used as the loading control, and pCR2.1-TOPO vector served as the internal control. A representative of three independent experiments is shown. G, IL-18-induced CXCL16 expression was not due to increased mRNA stability. Quiescent ASMC were treated with 25 ng/ml IL-18 for 2 h followed by the addition of 5 g/ml actinomycin D for up to 6 h. Total RNA was isolated and analyzed for CXCL16 expression by Northern blotting as before. Band intensity was determined by densitometry and normalized to the value obtained at 2 h (100%). The results are the mean S.E. of four independent experiments.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Expressing, Northern Blot, Isolation, Cell Culture, Control, Western Blot, Plasmid Preparation

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 2. IL-18-mediated CXCL16 expression is independent of IL-1, TNF-, and IFN-. A, quiescent ASMC were transfected with 10 M IL-1, TNF-, or IFN- antisense oligonucleotides and incubated for 16 h before the addition of 25 ng/ml IL-18. Cells were harvested at 2 h, and total RNA was isolated and analyzed for CXCL16 expression by Northern blotting. A representative of three independent experiments is shown. B, to exclude the role of preformed cytokines in IL-18-mediated CXCL16 expression, quiescent ASMC were incubated with IL-1-, TNF-, or IFN- neutralizing antibodies (5 g/ml) followed by the addition of 25 ng/ml IL-18 for 2 h, and CXCL16 expression was analyzed as in A. C and D, to confirm that the IL-18 induction of CXCL16 expression was independent of IFN-, ASMC were transfected with either IFN- or control siRNAs and incubated for 48 h. The cells were then untreated or treated with 25 ng/ml IL-18 for 3 h before isolation of total cell protein. Knock-down of IL-18-induced IFN- protein expression was confirmed by Western blotting (C, fifth lane). Knock-down of IFN- had no effect on the induction of CXCL16 mRNA by IL-18 (D, fifth lane).

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Expressing, Transfection, Incubation, Isolation, Northern Blot, Control, Knockdown, Western Blot

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 3. Genomic organization and potential regulatory elements of rat CXCL16 gene. A, the CXCL16 gene is located on rat chromosome 10 and is composed of 5 exons (boxes, roman numerals) and 4 introns (straight lines, Arabic numbers and circled). The positions of the ATG start codon and the TAG stop codon are indicated. Regions of the exons encoding the open reading frame are shown in black. The lengths of the exonic

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques:

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 4. IL-18 induces AP-1 DNA binding activity and AP-1-dependent reporter activity. A, IL-18 induces AP-1 DNA binding activity in ASMC. EMSA for AP-1 DNA binding activity was carried out using the AP-1 DNA binding sequence from the CXCL16 promoter, and nuclear extracts from quiescent ASMC or ASMC treated with 25 ng/ml IL-18 for the indicated times. Competition experiments (lanes 1 and 2) were performed with nuclear protein extracts from ASMC treated with IL-18 for 1 h. Arrows denote specific DNA-protein complexes. B, supershift assays. Nuclear extracts from ASMC treated with IL-18 for 1 h were incubated with anti-c-Fos, -c-Jun, -JunD, -JunB, or isoform-specific control IgG antibodies, and EMSA was performed as in A. Supershifted complexes are indicated by the white arrow. C, chromatin immunoprecipitation analysis of the CXCL16 AP-1 binding site. Quiescent ASMC were untreated or treated with IL-18 for 1 h, then cross-linked chromatin was prepared and immunoprecipitated with or without antibodies (Ab) to c-Fos before amplification of the CXCL16 gene region containing the AP-1 site. No specific DNA was seen in the chromatin immunoprecipitated without specific antibody (lanes 1 and 3). Specific DNA sequences in the chromatin immunoprecipitated by c-Fos antibody are shown (lanes 2 and 4). Amplification of the input DNA is shown in lanes 5 and 6. D, IL-18 increases transactivation by AP-1. The pAP-1-Luc reporter plasmid was co-transfected with the control pRL-TK Renilla luciferase plasmid into ASMC, and after 24 h, the transfected cells were incubated with antibody to IL-18 or control IgG for 1 h followed by the addition of 25 ng/ml IL-18 for 7 h. Firefly and Renilla luciferase activities were determined, and the luciferase activity was normalized to the Renilla values. The data represent the mean S.E. of four independent experiments. *, p 0.001 (versus untreated); †, p 0.01 versus IL-18-treated transfected cells. E, the AP-1 site in the rat CXCL16 promoter mediates IL-18-induced transcription. The AP-1 site in the pCXCL16-S2 promoter construct was mutated by site-directed mutagenesis from TGACTcaAATCC to TGACTtgAATCC, and the wild-type and mutant constructs were transfected and assayed as in D. *, p 0.001 versus S2-mut AP-1. IRF, interferon regulatory factor. F, IL-18 increases transactivation by NF-B. The pNF-B-Luc reporter plasmid was transfected and assayed as in D. The data represent the mean S.E. of four independent experiments. *, p 0.001 (versus untreated); †, p 0.01 versus IL-18-treated pNF-B-1-Luc transfected cells.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Binding Assay, Activity Assay, Sequencing, Incubation, Control, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Plasmid Preparation, Transfection, Luciferase, Construct, Mutagenesis

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 5. c-Jun and c-Fos transacti- vate the CXCL16 promoter. A, ASMC were co-transfected with CXCL16 pro- moter-reporter construct S2 and pRL-TK together with either dn-c-Fos, c-Fos anti- sense oligonucleotides, or c-Jun antisense oligonucleotides. Empty vector, sense, and scrambled oligonucleotides served as controls. After 48 h the cells were stimu- lated with 25 ng/ml IL-18 for 7 h before assay. B, ectopic expression of c-Fos and c-Jun activates CXCL16 promoter-re- porter activity. ASMC were transfected with S2, and c-Fos, c-Jun, or c-Fos plus c-Jun expression vectors together with the pRL-TK control vector. After 48 h the transfected cells were incubated with 25 ng/ml IL-18 for 7 h. Cell lysates in both studies were analyzed for reporter gene activities as in Fig. 4. The data are the mean S.E. of 3–6 independent experi- ments. A, *, p 0.001 versus untreated control; †, p 0.05 versus IL-18. B: *, p 0.05; **, p 0.01 versus untreated and pcDNA3.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Transfection, Construct, Plasmid Preparation, Expressing, Activity Assay, Control, Incubation

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 8. IL-18 induced p38 MAPK, ERK, and JNK activation in ASMC. A, IL-18 induced p38 MAPK activity. Quiescent ASMC were treated with 25 ng/ml IL-18 for the indicated time periods, and Western blot analysis was carried out on 40 g of the whole cell homogenates using antibodies to p38 MAPK and phospho-p38 MAPK. C, control. B, IL-18 induced ERK activation. ASMC were treated with IL-18 as in A, and Western blot analysis was carried out using antibodies against ERK and phospho-ERK. C, IL-18 induced JNK activation. ASMC were treated with IL-18 as in A, and Western blot analysis was carried out using antibodies against JNK and phospho-JNK. D, IL-18 induced JNK kinase activity. ASMC were treated with IL-18 as in A and at the indicated time periods. Cell lysates were harvested and analyzed for JNK kinase activity as described under “Materials and Methods.” Phosphorylated c-Jun was detected by Western blot analysis using anti-phospho c-Jun (Ser63) antibody. E, SB203580 inhibited IL-18-induced p38 MAPK phosphorylation. ASMC were treated with 1 M SB203580 for 30 min before the addition of 25 ng/ml IL-18. Total and phospho-p38 MAPK levels were determined by Western blotting. DMSO, Me2SO. F, PD98059 inhibited IL-18-induced ERK phosphorylation. ASMC were treated with 10 mM PD98059 for 1 h before the addition of 25 ng/ml IL-18. Total and phospho-ERK levels were determined by Western blotting. G, SP600125 inhibited IL-18-induced JNK phosphorylation. ASMC were treated with 20 mM SP600125 for 30 min before the addition of 25 ng/ml IL-18. Total and phospho-JNK levels were determined by Western blotting. H, however, JNK, but not p38 MAPK or ERK, mediated IL-18-induced CXCL16 mRNA expression. ASMC were treated with the p38 MAPK inhibitor SB203580, the ERK inhibitor PD98059, or the JNK inhibitor SP600125 (as above) followed by the addition of 25 ng/ml IL-18 for 2 h. Total RNA was isolated and analyzed for CXCL16 mRNA expression by Northern blotting. 28 S rRNA served as an internal control. A representative blot from three independent experiments is shown.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Activation Assay, Activity Assay, Western Blot, Control, Phospho-proteomics, Expressing, Isolation, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 9. IL-18 induced CXCL16 expression in ASMC through MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling pathways. A, IL-18 increased AP-1-dependent reporter gene activity via MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling. ASMC were co- transfected with pAP-1-Luc and the pRL-TK transfection control vector together with dnMyD88, dnIRAK1, dnIRAK4, or dnTRAF6 expression vectors. After 48 h the cells were treated with 25 ng/ml IL-18 for 7 h, and the reporter gene activity was assayed as before. Results are the mean S.E. of four independent experiments. *, p 0.001 versus untreated cells; †, p 0.05 versus IL-18. B, IL-18 induced CXCL16 gene transcription activity via MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling. ASMC were transfected with the S2 CXCL16 promoter-reporter vector and either dnMyD88, dnIRAK1, dnIRAK4, or dnTRAF6 expression vectors or their vector controls. Luciferase activity was assayed as before. The results are the mean S.E. of six experiments. *, p 0.001 (versus untreated); †, p 0.05 versus IL-18. C, IL-18 induced CXCL16 mRNA expression via MyD88-, IRAK1-, IRAK4-, and TRAF6-dependent signaling. ASMC were transfected with dnMyD88, dnIRAK1, dnIRAK4, or dnTRAF6 and after 24 h treated with 25 ng/ml IL-18 for 2 h. Total RNA was isolated and analyzed for CXCL16 mRNA expression by Northern blotting as before.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Expressing, Protein-Protein interactions, Activity Assay, Transfection, Control, Plasmid Preparation, Luciferase, Isolation, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 10. IL-18 induced CXCL16 expression via PI3K and Akt. A, IL-18 increased AP-1-dependent reporter gene activity via PI3K and Akt. ASMC were transfected with an AP-1 reporter vector (pAP-1-Luc) and pRL-TK transfection control vector. In some cases the cells were also co-transfected with a dominant negative PI3K expression vector, the empty vector (pcDNA3.1), or Akt siRNA. After 48 h some cells were treated with PI3K inhibitor, Akt inhibitor, or vehicle (Me2SO (DMSO)) for 1 h then treated with 25 ng/ml IL-18 or vehicle for 7 h. Reporter genes were assayed as before. Results are the mean S.E. of four independent experiments. *, p 0.001 (versus untreated); †, p 0.05 versus IL-18. B, IL-18 induced CXCL16 promoter-reporter activity via PI3K and Akt. The experiments were carried out as in A but using the S2 construct. Results are the mean S.E. of six experiments. *, p 0.001 (versus untreated); †, p 0.05 versus IL-18. C, IL-18 induced CXCL16 mRNA expression via PI3K and Akt. Experimental conditions were as described for panel B, except that the cells were treated with 25 ng/ml IL-18 for 2 h before Northern blot analysis. A representative blot from three experiments is shown.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Expressing, Activity Assay, Transfection, Plasmid Preparation, Control, Dominant Negative Mutation, Construct, Northern Blot

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 11. IL-18 induced CXCL16 expression through Src and JNK. A, IL-18 increased AP-1-dependent reporter gene activity via Src and JNK. ASMC were transfected with pAP-1-Luc and pRL-TK and after 24 h treated with the Src kinase inhibitors PP1 and PP2, the JNK inhibitor SP600125, or antisense JNK oligonucleotides followed by 25 ng/ml IL-18 for 7 h. Reporter gene assays were carried out as before. The results are the mean S.E. of four experiments. *, p 0.001 (versus untreated); †, p 0.05 versus IL-18. DMSO, Me2SO. B, IL-18 induced CXCL16 promoter-reporter activity via Src and JNK. Experimental conditions were as in A, except that the S2 construct was used instead of pAP-1-Luc. The results are the mean S.E. of six experiments. *, p 0.001 (versus untreated); †, p 0.05 versus IL-18. C, IL-18 induced CXCL16 mRNA expression via Src and JNK. Experimental conditions were as in A, except that the cells were treated with 20 ng/ml IL-18 for 2 h. Northern analysis was carried out as before. D, SP600125 inhibited the IL-18-induced phosphorylation of c-Jun Ser63 and c-Jun Ser73 in ASMC. To confirm that the dose of JNK inhibitor used in A inhibited the IL-18 activation of c-Jun, ASMC were treated with 20 M SP600125 for 30 min, then 25 ng/ml IL-18 for 1 h, and whole cell lysates were assayed for total c-Jun, c-Jun Ser63, and c-Jun Ser73 by Western blot. -Actin was used as the cell loading control.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Expressing, Activity Assay, Transfection, Construct, Northern Blot, Phospho-proteomics, Activation Assay, Western Blot, Control

Journal: Journal of Biological Chemistry

Article Title: The Pro-atherogenic Cytokine Interleukin-18 Induces CXCL16 Expression in Rat Aortic Smooth Muscle Cells via MyD88, Interleukin-1 Receptor-associated Kinase, Tumor Necrosis Factor Receptor-associated Factor 6, c-Src, Phosphatidylinositol 3-Kinase, Akt, c-Jun N-terminal Kinase, and Activator Protein-1 Signaling

doi: 10.1074/jbc.m502586200

Figure Lengend Snippet: FIG. 12. IL-18 induced ASMC proliferation in a CXCL16-dependent manner. A, IL-18 failed to induce ASMC death. Quiescent ASMC were treated with 25 ng/ml IL-18 for 24 h. The levels of mono- and oligonucleosomal fragmented DNA in the cytoplasmic extracts were quantified by enzyme-linked immunosorbent assay (ELISA). SNAP (500 mM), a nitric oxide donor, was used as a positive apoptosis-inducing control. The data represent the mean S.E. of four experiments. *, p 0.001 versus untreated and IL-18-treated cells. B, cells treated in the identical manner were also analyzed for annexin V/PI staining. The data shown are the percentage of annexin V-positive and phosphatidylinositol-negative cells after the treatment and represent the mean S.E. of three experiments. C, IL-18 induced phosphorylation of Bad. Quiescent ASMC were treated with 25 ng/ml IL-18 for 4 h. Cell lysates were analyzed by Western blotting for total Bad and for phospho-Bad using an antibody that specifically recognizes the phosphorylated Ser136 residue. -Actin was used as the internal loading control. D, IL-18 failed to induce caspase-3 activation. Quiescent ASMC were treated with 25 ng/ml IL-18 for 16 h. Activation of caspase-3 was analyzed by Western blotting. SNAP was used as a positive control. E, IL-18 failed to activate PARP. Quiescent ASMC were treated with 25 ng/ml IL-18 for16 h, and activation of PARP was analyzed in nuclear protein extracts by Western blotting. SNAP was used as a positive control. These experiments were repeated three times with similar results. F, ASMC expressed CXCR6. 1 mg of poly(A) RNA from quiescent ASMC was analyzed for CXCR6 mRNA by reverse transcription-PCR. Lane 1, 100-bp ladder; lane 2, CXCR6 cDNA (indicated by an arrow); lane 3, reverse transcription negative control. G, IL-18 induced ASMC proliferation. Quiescent ASMC were treated with or without 100 M CXCL16 siRNA or scrambled control RNA for 48 h before the addition of 25 ng/ml IL-18. Cell proliferation was determined by [3H]thymidine incorporation. The data shown are the mean S.E. of six experiments. *, p 0.01 versus control siRNA; †, p 0.025 versus IL-18. H, CXCL16 siRNAs specifically inhibited IL-18-induced CXCL16 expression. To confirm that the CXCL16 siRNA in G specifically inhibited IL-18-induced CXCL16 expression, ASMC treated exactly as in G were assayed for CXCL16 protein by Western blotting.

Article Snippet: Goat anti-mouse CXCL16 antibodies (AF503) and antiIFN- antibodies (AF-585-NA) were obtained from R&D Systems.

Techniques: Enzyme-linked Immunosorbent Assay, Control, Staining, Phospho-proteomics, Western Blot, Residue, Activation Assay, Positive Control, Reverse Transcription, Negative Control, Expressing

Journal: International journal of oncology

Article Title: Evaluation of CXCL10 as a novel serum marker for predicting liver metastasis and prognosis in colorectal cancer.

doi: 10.3892/ijo.2011.1247

Figure Lengend Snippet: Figure 2. (a), Scattergram of serum CXCL10 in stage I-IV colorectal cancer patients and control subjects (p<0.0001). (b), Scattergram of serum CXCL10 subdivided by pathological T stage (p=0.0101). (c), Scattergram of serum CXCL10 subdivided by clinical H stage (p=0.0004). (d), The relationships between serum CXCL10 and serum CEA level (r=0.2853, p<0.0001).

Article Snippet: Primary anti-CXCL10 goat polyclonal antibody (clone AF976, R&D Systems, MN, USA) and anti-CXCR3 rabbit polyclonal antibody (clone ab8023, Abcam, Cambridge, UK) at a dilution of 1:25 were used for the labeled streptavidinbiotin method (LASB2 kit/HRP, Dako Cytomation).

Techniques: Control

Journal: International journal of oncology

Article Title: Evaluation of CXCL10 as a novel serum marker for predicting liver metastasis and prognosis in colorectal cancer.

doi: 10.3892/ijo.2011.1247

Figure Lengend Snippet: Figure 3. (a), Cancer specific survival curves, for 218 patients with colorectal cancer, according to serum CXCL10 level. Patients with higher serum CXCL10 had significantly poorer prognoses than those with lower serum CXCL10 levels (p<0.0001, log-rank test); cut-off value = 199 pg/ml. (b), Cancer specific survival curves for 163 patients in stage I-III with curative intent, according to serum CXCL10 level. Patients with higher serum CXCL10 also had significantly poorer prognoses than those with lower serum CXCL10 levels (p=0.0372, log-rank test); cut-off value = 153 pg/ml.

Article Snippet: Primary anti-CXCL10 goat polyclonal antibody (clone AF976, R&D Systems, MN, USA) and anti-CXCR3 rabbit polyclonal antibody (clone ab8023, Abcam, Cambridge, UK) at a dilution of 1:25 were used for the labeled streptavidinbiotin method (LASB2 kit/HRP, Dako Cytomation).

Techniques:

Journal: International journal of oncology

Article Title: Evaluation of CXCL10 as a novel serum marker for predicting liver metastasis and prognosis in colorectal cancer.

doi: 10.3892/ijo.2011.1247

Figure Lengend Snippet: Figure 4. CXCL10 protein expression was assessed using immunohistochemistry in normal mucosa with primary CRC (a), primary colorectal cancer (b) and liver metastasis tissue (c). Magnification, x40. Scale bar = 500 µm.

Article Snippet: Primary anti-CXCL10 goat polyclonal antibody (clone AF976, R&D Systems, MN, USA) and anti-CXCR3 rabbit polyclonal antibody (clone ab8023, Abcam, Cambridge, UK) at a dilution of 1:25 were used for the labeled streptavidinbiotin method (LASB2 kit/HRP, Dako Cytomation).

Techniques: Expressing, Immunohistochemistry

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 1. VCAM-1, P-selectin, sTNFR-1, and CXCL16 are elevated in the urine in spontaneous lupus nephritis in three different mouse models of lupus. The levels of the above molecules were determined by ELISA in 24-h urine samples obtained from 6-mo-old B6.Sle1.lpr, MRL.lpr, and NZM2410 lupus mice (all of which exhibited proteinuria and glomerulonephritis), as well as 6-mo-old B6 controls (A). Indicated p values per- tain to Student’s t test or Mann-Whitney U test compar- isons of B6 urine vs the other groups (, p 0.05; , p 0.01; , p 0.001). n 5 mice/group. For each urine sample depicted in Fig. 1A, the 24-h urine level of total protein was plotted against the 24-h urine levels of the four molecules (B). Typically, healthy B6 mice do not excrete 1 mg of protein over a 24-h period (18, 19).

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 2. Urinary VCAM-1, P-selec- tin, sTNFR-1, and CXCL16 are higher than the corresponding serum levels. Se- rum and urine levels of the above four molecules were determined by ELISA, us- ing samples from 2-mo-old B6, 2-mo-old MRL.lpr, and 6-mo-old MRL.lpr mice. In- dicated p values pertain to Student’s t test or Mann-Whitney U test comparisons of B6 urine vs the other groups (indicated be- low the x-axis), or young vs old MRL.lpr samples, as indicated above each plot (, p 0.05; , p 0.01; , p 0.001). n 12 mice/group.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 3. The urinary enrichment of VCAM-1 and CXCL16 is also noted in other strains with spontaneous lupus nephritis. Serum and urine levels of VCAM-1 and CXCL16 were determined by ELISA, using samples from 2- to 5-mo-old B6, B6.Sle1.lpr, and BXSB mice, as in- dicated. Displayed p values pertain to the Student t test or Mann-Whit- ney U test comparisons of young vs old mice (, p 0.05; , p 0.01; , p 0.001); n 6 mice/group.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Enzyme-linked Immunosorbent Assay

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 4. VCAM-1, TNFR-1, P-selectin, and CXCL16 are hyperex- pressed within the diseased kidneys in lupus. Renal cortical lysates were prepared from B6 control, as well as BXSB and MRL.lpr mice, aged 5–6 mo. The levels of the four molecules were determined by ELISA. Indicated p values pertain to Student’s t test or Mann-Whitney U test comparisons of B6 vs other strains (, p 0.05; , p 0.01; , p 0.001). n 6 mice/group. Group means are indicated by the horizontal bars.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Control, Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 5. CXCL16, p-selectin, sTNFR-1, and VCAM-1 may be ex- pressed in the kidneys. Immunostaining of mouse kidneys from 2- and 7-mo-old MRL.lpr mice was performed using Abs against the above four molecules. Shown figures are representative of the histology seen in five mice per group.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: Immunostaining

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 6. Urine VCAM-1, P-selectin, sTNFR-1, and CXCL16 are also elevated in human lupus nephritis. A, Spot urine samples from 38 SLE patients (Table I), 15 healthy adults, and 6 RA patients were assayed for the levels of VCAM-1, P-selectin, sTNFR-1, and CXCL16. Indicated p values pertain to Student’s t test or Mann-Whitney U test comparisons of the urinary levels of each molecule in patients against that in normal controls (indicated below the x-axis), or SLE vs RA, as indicated at the top (, p 0.05; , p 0.01; , p 0.001). Shown also are the correlation profiles of these urinary molecules (normalized to urine creatinine) against the corresponding urine protein-creatinine ratios (B) and SLEDAI scores (C). R: correlation coefficient.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: MANN-WHITNEY

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 7. Specificity/sensitivity ROC curves per- taining to the use of urinary VCAM-1 (A), CXCL16 (B), sTNFR-1 (C), and P-selectin (D) for discriminating SLE patients from normal controls. The data shown in Fig. 6 were used to generate the shown ROC curves, which plot the specificity and sensitivity profiles of the indicated uri- nary molecules in distinguishing SLE patients from nor- mal controls. Indicated are the corresponding AUC values.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques:

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 8. SLE patients with active ne- phritis exhibit the highest levels of urine VCAM-1, P-selectin, sTNFR-1, and CXCL16. Urine levels of the indicated molecules were assayed in SLE patients and normal controls. The SLE patients were divided into four groups: (1) inactive SLE (SLEDAI 0); (2) mild-active, non- renal SLE (SLEDAI 1–5, renal SLE- DAI 0); (3) severe-active, nonrenal SLE (SLEDAI 6; renal SLEDAI 0); (4) ac- tive renal SLE (renal SLEDAI 0). Indi- cated p values pertain to Student’s t test or Mann-Whitney U test comparisons of each subject group vs “mild-active, nonrenal SLE” (, p 0.05; , p 0.01; , p 0.001).

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: MANN-WHITNEY

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 9. Specificity/sensitivity ROC curves per- taining to the use of urinary VCAM-1 (A) and CXCL16 (B) for discriminating SLE patients with active renal dis- ease. The data shown in Fig. 8 were used to generate the shown ROC curves, which plot the specificity and sensi- tivity profiles of the indicated urinary molecules in dis- tinguishing SLE patients with active renal disease from all other SLE patients. Indicated are the corresponding AUC values.

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques:

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

Article Title: Elevated urinary VCAM-1, P-selectin, soluble TNF receptor-1, and CXC chemokine ligand 16 in multiple murine lupus strains and human lupus nephritis.

doi: 10.4049/jimmunol.179.10.7166

Figure Lengend Snippet: FIGURE 10. Urine vs serum levels of VCAM-1, P-selectin, sTNFR-1, and CXCL16 in human lupus. Serum levels of the above four molecules were also assayed in SLE patients with inactive SLE (SLEDAI 0; n 5, randomly selected from the patients listed in Ta- ble I) and in patients with “active SLE” (SLEDAI 12; n 12, randomly se- lected from the patients listed in Table I). Displayed are the absolute levels (left column) as well as the albumin- normalized values (middle column) for the four molecules assayed. Displayed in the right column are the urine-serum ratios of these four molecules, after normalization against the correspond- ing albumin levels. Indicated p values pertain to Student’s t test or Mann- Whitney U test comparisons of patients vs “normal” (indicated below the x-axis), or “inactive” vs “active” pa- tients, as indicated above each plot (, p 0.05; , p 0.01; , p 0.001).

Article Snippet: Kidney sections obtained from 2- or 6-mo-old mice were stained with the following primary Abs: goat anti-mouse CXCL16 Ab (no. AF503; R&D Systems), goat anti-mouse TNFR-1 Ab (no. BAF425; R&D Systems), goat anti-mouse VCAM-1 (no. BAF643; R&D Systems) Ab, or goat anti-mouse P-selectin Ab (no. SC-6941; Santa Cruz Biotechnology) or the respective isotype control Abs, and developed as described previously (21).

Techniques: MANN-WHITNEY