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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 1. Semiquantitative RT-PCR analysis for expression of CCL28, CCR10, and CCR3 in various mouse tissues. Total RNA samples were prepared from salivary glands, colon, appendix, small intestine with- out Payer’s patches, and Payer’s patches obtained from 12- to 16-wk-old BALB/c mice. RT-PCR was conducted as described in Materials and Methods. Representative results from three separate experiments are shown. Relative signal intensities obtained by normalization with G3PDH are shown in the lower panels as mean SD.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 2. Surface expression of CCR10 and CCR3 and migration to CCL28 of CD3B220low cells isolated from mouse parotid glands. A, Flow cytometric analysis. Single cells were prepared from parotid glands excised from 12- to 16-wk-old BALB/c mice. For detection of cells expressing CCR10, cells were first incubated with CCL27-Fc or control Fc. After washing, cells were incubated with biotin-labeled goat anti- human IgG. After washing, cells were stained with a cocktail of APC-labeled streptavidin, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. For detection of cells expressing CCR3, cells first were incubated with rabbit polyclonal anti-mCCR3 or control rabbit IgG. After washing, cells were incubated with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. Finally, cells were analyzed on FACSCalibur. Representative results from six independent experiments are shown. B, Chemotaxis assay. Single cells prepared from parotid glands were added to the inserts of Transwell plates, with lower wells containing medium without or with indicated concentrations of mCCL2, mCCL28, or mCCL27. After 4 h at 37°C, cells migrated into lower wells were harvested. Original cells and cells migrated into lower wells were stained with PE-labeled anti-CD3 and CyChrome-labeled anti-B220 and were analyzed by flow cytometry in the presence of a known number of counting beads. Filled bars, CD3B220low cells; open bars, CD3B220 cells. The data are mean SD from three separate experiments. C, Plasma cell morphology of CCR10-expressing cells. Single cells prepared from mouse parotid glands were incubated with CCL27-Fc or control Fc. After washing, cells were stained with FITC-labeled goat anti-human IgG. Cells were then placed on glass slides, fixed with methanol, and further stained with May-Gru¨nwald-Giemsa. Cells in the same fields were observed on a fluorescent microscope under UV (Ca) and visible (Cb) lights. No FITC-staining cells were seen by control Fc (data not shown). Magnification, 1000.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Expressing, Migration, Isolation, Incubation, Control, Labeling, Staining, Chemotaxis Assay, Cytometry, Clinical Proteomics, Microscopy

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 3. Immunohistochemistry and immunoelectron microscopy of CCL28 in human and mouse salivary glands. Periodate-lysine-4% parafor- maldehyde-prefixed frozen sections of human submandibular gland (A–C, J, and K), mouse parotid gland (D and G), mouse submandibular gland (E and F), and mouse sublingual gland (F and I) were stained with goat anti- human CCL28 (A, B, and J), goat anti-mouse CCL28 (D–F), or normal goat IgG (C, G–I, and K). Immunohistochemistry (A–I): scale bar, 100 m (A–C) and 20 m (D–I); , acinus; , duct. Immunoelectron microscopy (J and K): scale bar, 1 m; arrow, immunoreactive granule; Lu, lumen.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Immunohistochemistry, Immuno-Electron Microscopy, Staining

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 4. Exocrine secretion of CCL28. A, Measurement of CCL28 in whole and parotid saliva by ELISA. Whole saliva and parotid secretions were obtained from healthy adult donors. All assays were done in triplicate and mean values were calculated. For details, see Materials and Methods. Whole and parotid saliva samples obtained from the same donors were connected by lines. B, Immunoblot analysis for CCL28. Recombinant CCL28, parotid saliva samples (10 l), and mature milk samples (20 l) were loaded as indicated. For details, see Materials and Methods. Repre- sentative results from two separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Recombinant

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 5. Analysis of the amino acid sequence of human CCL28. A, Amino acid comparison of histatin-5 and CCL28-C. The zinc-binding mo- tifs HExxH and HExxxH are underlined. B, Hydrophobicity plot of hista- tin-5 and CCL28-C. C, A phylogenic tree of chemokines and antimicrobial peptides. MEC/CCL28 and CCL28-C are boxed.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Sequencing, Comparison, Binding Assay

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 6. Antimicrobial assay. CCL28, mCCL28, CCL27, CCL28-C, and histatin-5 were examined for antimicrobial activity against C. albicans, P. aeruginosa, and Streptococcus mutans by using the CFU assay. For details, see Materials and Methods. All assays were done in triplicate. Vertical bars indicate SD. Representative results from three separate experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 8. Flow cytometric analysis on CCL28 antimicrobial activity. C. albicans were treated with CCL28 as indicated. After washing, cells were stained with FITC-labeled annexin V for 10 min and were washed again. After addition of PI at 2 g/ml, cells were immediately analyzed on FACSCalibur. The representative results of three independent experiments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Staining, Labeling

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 9. Antimicrobial activity of CCL28 analyzed by scanning electron microscopy. C. albicans (A–F) was mock treated (A and D) or treated with 10 M CCL28 for 30 min (B and E) or 60 min (C and F). P. aeruginosa (G and H) and Streptococcus mutans (I and J) were mock treated (G and I) or treated with 10 M CCL28 for 2 h (H and J). Microbes were immobilized, dried, coated with 3-nm thick platinum-paradium, and observed by a scanning electron microscope. Scale bars: A, 2 m; D, 200 nm; and G, 300 nm.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Electron Microscopy, Microscopy

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

Article Title: CCL28 has dual roles in mucosal immunity as a chemokine with broad-spectrum antimicrobial activity.

doi: 10.4049/jimmunol.170.3.1452

Figure Lengend Snippet: FIGURE 7. Salt-sensitive antimicrobial activity of CCL28. The effects of NaCl concentrations on the antimicrobial activity of CCL28 against C. albicans and P. aeruginosa were examined by using CFU assay. For de- tails, see Materials and Methods. All assays were done in triplicate. Ver- tical bars indicate SD. Representative results from two separate experi- ments are shown.

Article Snippet: We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs.

Techniques: Activity Assay, Colony-forming Unit Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 2 Tumor-derived CCL28 recruits pericytes to promote vascular normalization in the tumor microenvironment

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 3 Tumor-derived CCL28 promotes the expression of angiopoietin-1 via CCR3 in pericytes

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques: Derivative Assay, Expressing

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 6 CCL28 is involved in bevacizumab-mediated vascular normalization

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Journal of experimental & clinical cancer research : CR

Article Title: Pericytes recruited by CCL28 promote vascular normalization after anti-angiogenesis therapy through RA/RXRA/ANGPT1 pathway in lung adenocarcinoma.

doi: 10.1186/s13046-024-03135-3

Figure Lengend Snippet: Fig. 7 A schematic diagram of tumor microenvironment modulation effects of CCL28

Article Snippet: Chromatin Immunoprecipitation (ChIP) Briefly, pericytes treated with or without recombinant human CCL28 or CCR3 (R&D Systems, MAB155-100) neutralizing antibodies were collected and fixed by adding a cross-linking agent, formaldehyde, to stabilize the interactions between chromatin proteins and DNA.

Techniques:

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 1. Analysis of chemokine and chemokine receptor mRNA levels in VEGFD- 293EBNA cells and VEGFD–treated LECs. A, Miniarrays containing oligonucleotides for chemokine and chemokine receptor genes were probed with biotin-labeled cRNA derived from VEGFD-293EBNA cells and human LECs. Signals for CCL27, CCL28, and CCR10 are indicated. B, Quantification of mRNA levels for CCL27 and CCL28 in VEGFD-293EBNA cells using qRT-PCR. Chemokine expression was normalized to b-actin. Data represent mean SEM of three replicates. C, qRT-PCR quantification of CCR10 expression in human LECs or BECs treated with 500 ng/mL VEGFD for 24 hours. Expression was normalized to b-actin. Data represent mean SEM of three replicates. , P < 0.05 by Student t test. D, Flow cytometry of LEC CCR10 cell surface expression using an anti-CCR10 antibody (dashed line) and isotype control (solid line). LECs were transfected with negative control (left) or CCR10_4 siRNA (right). E, Knockdown of CCR10 mRNA by four different siRNAs was assessed by qRT-PCR, compared with a negative control (normalized to 100%). Data represent mean SEM of three experiments. , P < 0.05 by Student t test. F, Serial tissue sections of human breast cancer (ductal carcinoma in situ, DCIS) and normal colon were immunostained for

Article Snippet: Chemokines, growth factors, and ELISA Recombinant human CCL27, CCL28, VEGFA, IFNg , TNFa, IL1b (all from R&D Systems), and mature VEGFC and VEGFD (Opthea Pty Ltd) were used in various assays.

Techniques: Labeling, Derivative Assay, Quantitative RT-PCR, Expressing, Flow Cytometry, Control, Transfection, Negative Control, Knockdown, In Situ

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 2. CCL27 and CCL28 protein localization in tumor-associated lymphangiogenesis. A, Apex-293EBNA, VEGFD-293EBNA, and FEMX-I xenograft tumors were stained via IHC with LYVE1, CCL27, or CCL28 antibodies. Top insets show the position of the main image (black box) in a lower magnification overview of the tumor section. Lower insets show a serial field of the main image at the same magnification stained with a corresponding isotype-matched negative control antibody. Scale bar, 100 mm in main; 200 mm in overview. B, Immunofluorescence staining of VEGFD-293EBNA tumors with LYVE1 and CCL27 antibodies. Scale bar, 100 mm. C, CCL28 staining on a lymphatic vessel in a FEMX-I tumor section. Scale bar, 100 mm. D, Relative expression of CCR10 and CCL27 mRNA in samples of human melanoma (n ¼ 45), benign nevi (n ¼ 18), and normal skin (n ¼ 7) from the publicly available microarray expression dataset GSE3189 (20). Data are presented as individual points with mean SEM (, P < 0.05; , P < 0.001; , P < 0.0001 by Mann–Whitney test).

Article Snippet: Chemokines, growth factors, and ELISA Recombinant human CCL27, CCL28, VEGFA, IFNg , TNFa, IL1b (all from R&D Systems), and mature VEGFC and VEGFD (Opthea Pty Ltd) were used in various assays.

Techniques: Staining, Negative Control, Expressing, Microarray, MANN-WHITNEY

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 4. CCL27, CCL28, and CCR10 mediate LEC migration but not proliferation in vitro. A, Proliferation assay of LECs stimulated with 0–500 ng/mL of recombinant CCL27, CCL28, or VEGFD. Proliferation was compared with unstimulated controls. Data represents mean SEM of three replicates. , P < 0.05 by Student t test. B, LECs were assessed for their ability to migrate toward titrated concentrations of CCL27, CCL28, VEGFC, or VEGFD in a transwell migration chamber over 21–24 hours. Migrated cells were quantified and compared with migration toward basal medium alone (Starve). Complete growth medium (Complete) was used as a positive control. Data represent mean SEM of three replicates; concentrations of each chemokine or VEGF (0.001–500 ng/mL) were compared with Starve control using a one-way ANOVA with Dunnett post hoc test. , P < 0.05; , P < 0.001; , P < 0.0001. C, BECs (left) and LECs (right) were assessed for chemotactic migration toward the indicated factors over 21–24 hours, as compared with migration toward starvation medium alone. Data represents mean SEM of three replicates; results compared using one-way ANOVA with Tukey's post hoc test; chemokines and VEGFs (positive controls) analyzed separately. , P < 0.05; , P < 0.01. D, Migration of LECs toward CCL27, CCL28, or VEGFA after CCR10 knockdown with siRNA. Data represents mean SEM of two technical replicates. , P < 0.05 by Student t test.

Article Snippet: Chemokines, growth factors, and ELISA Recombinant human CCL27, CCL28, VEGFA, IFNg , TNFa, IL1b (all from R&D Systems), and mature VEGFC and VEGFD (Opthea Pty Ltd) were used in various assays.

Techniques: Migration, In Vitro, Proliferation Assay, Recombinant, Positive Control, Control, Knockdown

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 5. CCL28 and CCL27 cooperate with VEGFD to attract LECs in vivo. A–E, FVB mice were injected subcutaneously with 200 mL Matrigel containing PBS (A) or 2 mg/mL of recombinant human CCL27 (B), 4 mg/mL VEGFD (C), 2 mg/mL CCL27 and 4 mg/mL VEGFD (D), or 4 mg/mL VEGFA (E). Matrigel plugs were harvested after one week and sections thereof stained with antibodies to LYVE1 (green) and PECAM1 (red) and nuclei counterstained with DAPI (blue). Scale bars, 100 mm. F and G, Quantification of LYVE1-positive vessels (F) or PECAM1-positive vessels (G) in Matrigel plugs. Data points represent average % stained area of <15 fields per mouse with mean SEM of n ¼ 6 mice per group (5 for PBS group); P values from Kruskal–Wallis test with uncorrected Dunn test. , P < 0.05; , P < 0.01; , P < 0.001. H and I, A similar experiment was conducted in which 2.5 mg CCL28 and VEGFD, alone or in combination as shown, were injected subcutaneously into FVB mice in 500 mL of Matrigel. LYVE1 staining was quantified in H and PECAM1 staining in I. Data points represent mean average % stained area of <15 fields per mouse with mean SEM of n ¼ 4 mice (3 for PBS group); P values from Kruskal–Wallis test with uncorrected Dunn test; , P < 0.05; , P < 0.01. J, CCL28 Matrigel plug costained with antibodies to LYVE1 (green) and podoplanin (red). Right, magnified view of the boxed area. Scale bars, 50 mm. K, CCL28 Matrigel plug costained with LYVE1 (green) and the macrophage marker F4/80 (red) showing infiltration of macrophages into the plug. Right, a magnified view of the boxed area. Open arrows, LYVE1þ LECs; closed arrows, F4/80þ macrophages. Scale bars, 50 mm.

Article Snippet: Chemokines, growth factors, and ELISA Recombinant human CCL27, CCL28, VEGFA, IFNg , TNFa, IL1b (all from R&D Systems), and mature VEGFC and VEGFD (Opthea Pty Ltd) were used in various assays.

Techniques: In Vivo, Injection, Recombinant, Staining, Marker

Journal: Cancer Research

Article Title: CCL27/CCL28–CCR10 Chemokine Signaling Mediates Migration of Lymphatic Endothelial Cells

doi: 10.1158/0008-5472.can-18-1858

Figure Lengend Snippet: Figure 7. Schematic diagram of mechanisms by which lymphangiogenic growth factors and chemokines may promote tumor spread. We and others have shown that CCL28 and/or CCL27 expression in tumor cells (blue cells) and CCR10 in LECs may be upregulated by TNFa (green arrows). TNFa and other inflammatory cytokines may be derived from macrophages recruited by tumor-secreted VEGFD, or by other cells in the tumor microenvironment. CCR10 can also be upregulated in LECs by VEGFD signaling via VEGF receptors VEGFR3 or VEGFR2 (green arrow), thus potentiating migration of LECs toward CCL27 or CCL28. Our data suggest a model whereby CCR10 upregulation may be polarized in particular migratory LECs within sprouting lymphatic vessels, with CCR10 and VEGFR3 signaling potentially operating in the same cells or adjacent cells. While CCL27 and CCL28 can guide LEC migration, cooperation with VEGFD signaling through VEGF receptors is required to support LEC proliferation and remodeling to generate lymphatic vessels that can support metastatic spread of tumor cells.

Article Snippet: Chemokines, growth factors, and ELISA Recombinant human CCL27, CCL28, VEGFA, IFNg , TNFa, IL1b (all from R&D Systems), and mature VEGFC and VEGFD (Opthea Pty Ltd) were used in various assays.

Techniques: Expressing, Derivative Assay, Migration

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

Article Title: Epithelial inflammation is associated with CCL28 production and the recruitment of regulatory T cells expressing CCR10.

doi: 10.4049/jimmunol.177.1.593

Figure Lengend Snippet: FIGURE 1. Livers from patients with chronic inflammatory liver diseases, including PSC, PBC, and ALD are heavily infiltrated by CD3 lymphocytes. a, T cells are centered on inflamed bile ducts (arrow). Immunohistochemistry revealed intense CCL28 staining (detected as a brown pigment) on inflamed bile ducts (B) and to a lesser extent on portal vein (PV) and hepatic artery (A) endothelium. b, Little staining was seen on normal liver or spleen. Control sections had no detectable staining. We confirmed our observations with Western blotting (c), which demonstrated minimal CCL28 protein in normal liver and spleen but enhanced expression in chronic liver disease. Sample loading was normalized for -actin and band intensity quantified using a Gel Doc system. Real-time RT-PCR of total liver mRNA samples confirmed increased CCL28 mRNA in diseased liver (p 0.001, Student’s t test) but also normal liver (p 0.006, Student t test) compared with skin after correction for 18S mRNA levels. d, Samples consisted of tissue samples from six donors each for PSC, PBC, normal liver, and ALD and three donors of spleen and skin tissue samples. Representative immunohistochemistry sections and Western blot results are shown. Results are expressed as mean SEM. , p 0.006 , p 0.001.

Article Snippet: The following primary Abs were used: mouse anti-human CCL28 mAb (62705, 5 g/ml; R&D Systems), mouse anti-human 4 7 (ACT-1, 15 g/ml; a gift from M. Briskin (Millennium Pharmaceuticals, Cambridge, MA), CD31 mAb (JC70A, 1 g/ml; DakoCytomation), cytokeratin-19 mAb (BA17, 1 g/ml; DakoCytomation), MAdCAM-1 mAb (CA 102.2C1, 12.5 g/ml; Serotec), mouse anti-human anti- 1 mAb (3S3, 4 g/ml; Abcam), mouse anti-human mAb HEA-125 (Progen), mouse antihuman 7-Cy5 mAb (FIB504, 1/20; BD Pharmingen), goat anti-human CCR10 (ab3904, 0.05 g/ml; Abcam), CD8-FITC mAb (DK25; 1/50; DakoCytomation), CD4-PE-Cy5 mAb (C7069; 1/50; DakoCytomation), CD3 R-PE-Cy7 mAb (UCHT1, 1/20; Beckman Coulter), CD25-FITC (F0801, 1/50; DakoCytomation), CD56-PE mAb (MOC-1, 1/30; DakoCytomation), CD104-FITC mAb (Ber-ACT8, 1/25; DakoCytomation), CD49d-FITC mAb (P4G9, 1/50; DakoCytomation), CCR7 mAb (fab197p, 1/20; R&D Systems), and CXCR3 mAb (fab160p, 1/20; R&D Systems).

Techniques: Immunohistochemistry, Staining, Control, Western Blot, Expressing, Quantitative RT-PCR

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

Article Title: Epithelial inflammation is associated with CCL28 production and the recruitment of regulatory T cells expressing CCR10.

doi: 10.4049/jimmunol.177.1.593

Figure Lengend Snippet: FIGURE 2. Coimmunofluorescence was used to confirm the cellular distribution of CCL28. Abs to CCL28 labeled with FITC green colocalized with anti-CD31 mAb labeled with Texas Red (TxRED) on portal endo- thelium (upper; yellow) product. There was little CCL28 expression on sinusoidal endothelium (middle). The strongest staining of CCL28 was detected on bile ducts and where anti-CCL28 labeled with FITC colocal- ized with an Ab against the epithelial Ag HEA labeled with Texas Red (lower). The staining shown on tissue sections from a patient with PSC. The findings are representative of the patterns of immunofluorescence staining seen in tissue sections from six donors each for PSC, PBC, and ALD. Sections stained with control Abs demonstrated minimal background tissue fluorescence.

Article Snippet: The following primary Abs were used: mouse anti-human CCL28 mAb (62705, 5 g/ml; R&D Systems), mouse anti-human 4 7 (ACT-1, 15 g/ml; a gift from M. Briskin (Millennium Pharmaceuticals, Cambridge, MA), CD31 mAb (JC70A, 1 g/ml; DakoCytomation), cytokeratin-19 mAb (BA17, 1 g/ml; DakoCytomation), MAdCAM-1 mAb (CA 102.2C1, 12.5 g/ml; Serotec), mouse anti-human anti- 1 mAb (3S3, 4 g/ml; Abcam), mouse anti-human mAb HEA-125 (Progen), mouse antihuman 7-Cy5 mAb (FIB504, 1/20; BD Pharmingen), goat anti-human CCR10 (ab3904, 0.05 g/ml; Abcam), CD8-FITC mAb (DK25; 1/50; DakoCytomation), CD4-PE-Cy5 mAb (C7069; 1/50; DakoCytomation), CD3 R-PE-Cy7 mAb (UCHT1, 1/20; Beckman Coulter), CD25-FITC (F0801, 1/50; DakoCytomation), CD56-PE mAb (MOC-1, 1/30; DakoCytomation), CD104-FITC mAb (Ber-ACT8, 1/25; DakoCytomation), CD49d-FITC mAb (P4G9, 1/50; DakoCytomation), CCR7 mAb (fab197p, 1/20; R&D Systems), and CXCR3 mAb (fab160p, 1/20; R&D Systems).

Techniques: Labeling, Expressing, Staining, Control

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

Article Title: Epithelial inflammation is associated with CCL28 production and the recruitment of regulatory T cells expressing CCR10.

doi: 10.4049/jimmunol.177.1.593

Figure Lengend Snippet: FIGURE 3. To determine factors that induce CCL28 expression, we isolated and cultured primary BEC from human livers (a) and stimulated confluent highly pure cultures for 24 h with combinations of TNF-, IFN-, TNF-, IL-1 (all 10 ng/ml), LPS (1 g/ml), or 500 M of the bile acid CDCA. CCL28 protein was measured by sandwich ELISA (b) and mRNA by real-time PCR (c). LPS and IL-1 were the most potent inducers of CCL28 expression (, p 0.002). Treatment with CDCA also induced CCL28 secretion (, p 0.01), but at a lower level than LPS and IL-1 treatment. Experiments reflect BEC isolated from six donors (n 3 PSC, n 1 PBC, n 2 ALD) and assays conducted in triplicate. Results are expressed as mean SEM. , p 0.01 , p 0.002 by Student t test compared with unstimulated cultures.

Article Snippet: The following primary Abs were used: mouse anti-human CCL28 mAb (62705, 5 g/ml; R&D Systems), mouse anti-human 4 7 (ACT-1, 15 g/ml; a gift from M. Briskin (Millennium Pharmaceuticals, Cambridge, MA), CD31 mAb (JC70A, 1 g/ml; DakoCytomation), cytokeratin-19 mAb (BA17, 1 g/ml; DakoCytomation), MAdCAM-1 mAb (CA 102.2C1, 12.5 g/ml; Serotec), mouse anti-human anti- 1 mAb (3S3, 4 g/ml; Abcam), mouse anti-human mAb HEA-125 (Progen), mouse antihuman 7-Cy5 mAb (FIB504, 1/20; BD Pharmingen), goat anti-human CCR10 (ab3904, 0.05 g/ml; Abcam), CD8-FITC mAb (DK25; 1/50; DakoCytomation), CD4-PE-Cy5 mAb (C7069; 1/50; DakoCytomation), CD3 R-PE-Cy7 mAb (UCHT1, 1/20; Beckman Coulter), CD25-FITC (F0801, 1/50; DakoCytomation), CD56-PE mAb (MOC-1, 1/30; DakoCytomation), CD104-FITC mAb (Ber-ACT8, 1/25; DakoCytomation), CD49d-FITC mAb (P4G9, 1/50; DakoCytomation), CCR7 mAb (fab197p, 1/20; R&D Systems), and CXCR3 mAb (fab160p, 1/20; R&D Systems).

Techniques: Expressing, Isolation, Cell Culture, Sandwich ELISA, Real-time Polymerase Chain Reaction

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

Article Title: Epithelial inflammation is associated with CCL28 production and the recruitment of regulatory T cells expressing CCR10.

doi: 10.4049/jimmunol.177.1.593

Figure Lengend Snippet: FIGURE 5. LIL express functional CCR10. a, The ability of CCL28 to activate lymphocyte binding to MAdCAM-1 and VCAM-1 was studied using total T lymphocyte populations from diseased livers (n 6). Treat- ment of LIL with either 10 ng or 50 ng/ml CCL28 significantly increased adhesion to both VCAM-1 and MAdCAM-1. Adhesion was inhibited by preincubation of lymphocytes with pertussis toxin (ptx) or blocking mAbs to either 1 or 47 integrins. Manganese (Mn) was used as a positive control as a nonsignaling activator of integrin adhesion. b, Migration of LIL to CCL28 was assessed using fibronectin-coated Transwell migration chambers. CXCL12 and BSA were use as controls. A total of 5 105

Article Snippet: The following primary Abs were used: mouse anti-human CCL28 mAb (62705, 5 g/ml; R&D Systems), mouse anti-human 4 7 (ACT-1, 15 g/ml; a gift from M. Briskin (Millennium Pharmaceuticals, Cambridge, MA), CD31 mAb (JC70A, 1 g/ml; DakoCytomation), cytokeratin-19 mAb (BA17, 1 g/ml; DakoCytomation), MAdCAM-1 mAb (CA 102.2C1, 12.5 g/ml; Serotec), mouse anti-human anti- 1 mAb (3S3, 4 g/ml; Abcam), mouse anti-human mAb HEA-125 (Progen), mouse antihuman 7-Cy5 mAb (FIB504, 1/20; BD Pharmingen), goat anti-human CCR10 (ab3904, 0.05 g/ml; Abcam), CD8-FITC mAb (DK25; 1/50; DakoCytomation), CD4-PE-Cy5 mAb (C7069; 1/50; DakoCytomation), CD3 R-PE-Cy7 mAb (UCHT1, 1/20; Beckman Coulter), CD25-FITC (F0801, 1/50; DakoCytomation), CD56-PE mAb (MOC-1, 1/30; DakoCytomation), CD104-FITC mAb (Ber-ACT8, 1/25; DakoCytomation), CD49d-FITC mAb (P4G9, 1/50; DakoCytomation), CCR7 mAb (fab197p, 1/20; R&D Systems), and CXCR3 mAb (fab160p, 1/20; R&D Systems).

Techniques: Functional Assay, Binding Assay, Blocking Assay, Positive Control, Migration

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 1. Expression of CCL28 in the porcine uterine endometrium during the estrous cycle and pregnancy. A) Real-time RT-PCR analysis of CCL28 mRNA in the uterine endometrium. Endometrial tissue samples from cyclic and pregnant gilts were analyzed by real-time RT-PCR, and the data are reported as expression relative to that detected on Day 12 of the estrous cycle after normalization of the transcript amount to the endogenous RPL7 control. B) In situ hybridization analysis of CCL28 mRNA in the uterine endometrium. Expression of CCL28 mRNA was localized mainly to endometrial GE cells during the estrous cycle and pregnancy. Representative uterine sections from Day 12 of pregnancy, hybridized with a DIG-labeled sense CCL28 cRNA probe (Sense) as a negative control, are shown. D, day; C, estrous cycle; P, pregnancy; LE, luminal epithelium; GE, glandular epithelium; St, stroma; BV, blood vessels. Bars ¼ 200 lm and 100 lm in inset. C) Immunoblot analysis of the CCL28 protein in uterine flushings on Day 12 of the estrous cycle and pregnancy. Uterine flushings were obtained from Day (D) 12 of the estrous cycle (C) and pregnancy (P) and the presence of the CCL28 protein was determined. We loaded rCCL28 as a positive control.

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Expressing, Quantitative RT-PCR, Control, In Situ Hybridization, Labeling, Negative Control, Western Blot, Positive Control

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 4. Expression of CCR3 and CCR10 in conceptuses and expression of CCR10 in chorioallantoic membranes. A) RT-PCR analyses of CCL28, CCR3, and CCR10 mRNAs in the uterine endometrium and conceptuses at Days 12 and 15 of pregnancy. Expression of CCL28 and CCR10, but not CCR3, mRNAs was detectable in conceptuses on Days 12 and 15 of pregnancy. RPL7 was used as a positive control. RTase þ/, with (þ) or without () reverse transcriptase; M, molecular marker; D12 Endo, endometrium from Day 12 of pregnancy; D15 Endo, endometrium from Day 15 of pregnancy; D12 Con, Day 12 conceptus; D15 Con, Day 15 conceptus. B) Immunohistochemical analysis of the CCR10 protein in a Day 12 conceptus. CCR10 protein was detected in conceptus trophec- toderm derived from Day 12 of pregnancy. D12P, Day 12 of pregnancy; Tr, trophectoderm; En, endoderm. Bar¼ 50 lm. C) Real-time RT-PCR analysis of the expression of CCR10 mRNA in porcine chorioallantoic tissues during pregnancy. Analysis of the expression of CCR10 mRNA in chorioallantoic tissue samples on Days 30, 60, 90, and 114 of pregnancy revealed that the expression of CCR10 mRNA increased during late pregnancy (linear effect of day, P , 0.01). The data are reported as expression relative to that detected on Day 30 of pregnancy after normalization of the transcript amount to the endogenous RPL7 control, and are presented as least-squares means with SEM.

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Positive Control, Reverse Transcription, Marker, Immunohistochemical staining, Derivative Assay, Quantitative RT-PCR, Control

Journal: Biology of reproduction

Article Title: Chemokine (C-C motif) Ligand 28 and Its Receptor CCR10: Expression and Function at the Maternal-Conceptus Interface in Pigs.

doi: 10.1095/biolreprod.116.141903

Figure Lengend Snippet: FIG. 5. Effect of CCL28 on pTr cell proliferation and migration. A) RT-PCR analysis of CCR3 and CCR10 mRNAs in the pTr cell line. The pTr cell line and PBMC as a control expressed CCR10 mRNA, but pTr cells did not express CCR3 mRNA. RPL7 was used as a positive control for RT-PCR. RTase þ/, with (þ) or without () reverse transcriptase; M, molecular marker; D12 Endo, endometrium from Day 12 of pregnancy. B) Effect of rCCL28 on pTr cell proliferation. After serum starvation for 24 h, pTr cells were treated with 0, 0.1, 1, 10, or 100 ng/ml rCCL28 at 378C for 48 h in triplicate. C) Effect of rCCL28 on pTr cell migration. Serum-starved pTr cells were seeded on 8-lm-pore Transwell inserts and treated with different doses (0, 10, or 100 ng/ml) of rCCL28 or 1 lg/ml of rSPP1 as a positive control in triplicate for 12 h. Unmigrated cells on the upper side of the inserts were removed, and cells that had migrated were counted systematically in five nonoverlapping locations. Each independent experiment for cell proliferation and migration was replicated three times. The asterisks denote statistically significant differences (*P , 0.05; **P , 0.01).

Article Snippet: Fifty-five micrograms of protein from concentrated uterine flushings and 0.5 lg of recombinant human CCL28 protein (rCCL28; R&D Systems) as a control were loaded into and electrophoresed on 12% SDSPAGE gels followed by electrotransfer onto nitrocellulose membranes.

Techniques: Migration, Reverse Transcription Polymerase Chain Reaction, Control, Positive Control, Reverse Transcription, Marker

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022X.2024.2313171

Figure Lengend Snippet: Patients’ clinical characteristics.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques: Control

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022X.2024.2313171

Figure Lengend Snippet: Plasma and urine CCL2 levels between AKI and non-AKI patients at hospital and ICU admission. ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques: Clinical Proteomics

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022X.2024.2313171

Figure Lengend Snippet: The predictive performance of urine CCL2 for AKI by ROC analysis. (A) ROC curve. (B) AUC and prediction sensitivity and specificity.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques:

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022X.2024.2313171

Figure Lengend Snippet: Plasma and urine CCL2 levels between SAKI and non-septic AKI patients at hospital and ICU admission. *** p < 0.001.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques: Clinical Proteomics

Journal: Renal Failure

Article Title: Assessment of urine CCL2 as a potential diagnostic biomarker for acute kidney injury and septic acute kidney injury in intensive care unit patients

doi: 10.1080/0886022X.2024.2313171

Figure Lengend Snippet: The predictive performance of urine CCL2 for SAKI by ROC analysis. (A) ROC curve. (B) AUC and prediction sensitivity and specificity.

Article Snippet: The CCL2 levels in plasma and urine were measured using a commercial human CCL2 ELISA kit (R&D Systems, Bio-Techne, Minneapolis, USA) following the manufacturer’s instructions.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: Western blot of AR in THP-1 scramble (scr) and silenced AR (siAR) cells. Migration assay of LNCaP/THP-1 scr and LNCaP/THP-1 siAR cells co-cultured for 24 h. Schematic illustration of LNCaP/THP-1 co-culture is shown, ( n = 3); bar in graph, Mean ± SEM; bars in pictures, 400 μm (magnification is 100×). Proliferation assay of LNCaP alone, LNCaP/THP-1 scr, or LNCaP/THP-1 siAR cells co-cultured for 24, 48 and 72 h, ( n = 3). Cytokine array of different conditioned media (CM) of LNCaP and THP-1 cells. CM of LNCaP, THP-1, LNCaP/THP-1 scr and LNCaP/THP-1 siAR cells were collected after 24 h incubation. CCL2 showed the most obvious increase in co-cultured CM of LNCaP/THP-1 siAR among these four sets (yellow squares). Cytokine array of different CM of C4-2 and THP-1 cells. CM of C4-2 scr/THP-1 scr and C4-2 siAR/THP-1 siAR cells were collected after 24 h incubation. CCL2 showed obvious increase in co-cultured CM of C4-2 siAR/THP-1 siAR cells (yellow squares, lower panel). Western blot analysis of AR expression in C4-2 scr and siAR cells (upper panel).

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Western Blot, Migration, Cell Culture, Co-Culture Assay, Proliferation Assay, Incubation, Expressing

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: qPCR of CCL2 mRNA in THP-1 scramble (scr) and THP-1 silenced AR (siAR) cells/different PCa cell lines as indicated ( left ) and qPCR of CCL2 mRNA in C4-2 scr and C4-2 siAR cells ( right ). qPCR of CCL2 mRNA in THP-1 (scr or siAR) cells co-cultured with C4-2 scr or siAR cells ( left ) and in C4-2 (scr or siAR) cells co-cultured with THP-1 scr or siAR cells ( right ). ELISA of CCL2 in 24 h CM of C4-2 scr and C4-2 siAR cells ( left ) and in 24 h co-cultured CM of C4-2 scr or C4-2 siAR cells/THP-1 scr or siAR cells ( right ). ELISA of CCL2 in 24 h co-cultured CM of parental LNCaP cells/THP-1 scr or siAR cells ( left ) and in 24 h co-cultured CM of parental LAPC4 cells/THP-1 scr or siAR cells ( right ). Migration assay of C4-2 scr and C4-2 siAR cells incubated for 24 h ( upper left ), parental THP-1 cells/C4-2 scr or siAR cells after co-cultured for 16 h ( upper right ), parental C4-2 cells/THP-1 scr cells or siAR cells after co-cultured for 24 h ( lower left ), and C4-2 scr or C4-2 siAR cells/THP-1 scr or siAR cells after co-cultured for 24 h ( lower right ), ( n = 3); bars in graphs (A–E), Mean ± SEM; bars in pictures, 400 μm (magnification 100×). Western blot of CCL2, EMT markers, AR, and PSA in parental C4-2 cells treated with CM of THP-1 scr and siAR, or co-cultured with THP-1 scr and siAR cells for 24 h ( left ), and in C4-2 scr and siAR cells ( right ).

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Migration, Incubation, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: Neutralization of CCL2 in migration assay of C4-2 scramble (scr) and AR silenced (siAR) cells incubated for 24 h. Neutralization of CCL2 in migration assay of parental THP-1 cells + C4-2 scr or +C4-2 siAR cells co-cultured for 16 h. Neutralization of CCL2 in migration assay of parental C4-2 cells + THP-1 scr or +THP-1 siAR cells co-cultured for 24 h. Neutralization of CCL2 in migration assay of C4-2 scr and C4-2 siAR cells + THP-1 scr or +THP-1 siAR cells co-cultured for 24 h. Anti-CCL2 antibody (30 µg/ml; CCL2ab) and mouse IgG (control) were used in A–D. ( n = 3); bars in graphs, Mean ± SEM in (A–D); bars in pictures, 400 μm (magnification 100×, A, C and D). Western blots of EMT markers (including the zymography of MMP9) in C4-2 scr and siAR cells incubated for 24 h with or without CCL2ab.

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Neutralization, Migration, Incubation, Cell Culture, Control, Western Blot, Zymography

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: qPCR of CCR2 in C4-2 scramble (scr) cells co-cultured with or without THP-1 scr cells and C4-2 AR silenced (siAR) cells co-cultured with or without THP-1 siAR cells for 24 h. Neutralization of CCR2 in migration assay of parental THP-1 cells + C4-2 siAR cells co-cultured for 16 h. Neutralization of CCR2 in migration assay of C4-2 siAR cells + THP-1 siAR cells co-cultured for 24 h. We used the same concentration of anti-CCL2 antibody (CCL2ab) in and 20 nM CCR2 antagonist (CCR2atg) diluted with DMSO used as treatment and DMSO used as control in (B and C), ( n = 3); bars in graphs, Mean ± SEM in (A–C); bars in pictures, 400 μm (magnification 100×, C). Proliferation assay of parental C4-2, C4-2 scr and C4-2 siAR cells incubated for 24, 48 and 72 h. Proliferation assay of parental C4-2 cells + parental THP-1, +THP-1 scr, or +THP-1 siAR cells co-cultured for 24, 48 and 72 h. Proliferation assay of C4-2 scr and C4-2 siAR cells + THP-1 scr or +THP-1 siAR cells co-cultured for 24, 48 and 72 h. Neutralization of CCL2 in proliferation assay of C4-2 siAR cells + THP-1 siAR cells co-cultured for 24, 48 and 72 h. 30 µg/ml CCL2ab and mouse IgG (control) were used. Neutralization of CCR2 in proliferation assay of C4-2 siAR cells + THP-1 siAR cells co-cultured for 24, 48 and 72 h. 30 µg/ml CCL2ab and 20 nM CCR2atg diluted with DMSO were used as treatment, ( n = 3); bars in graphs, Mean ± SEM in (D–H). Western blots of STAT3 and EMT markers in C4-2 scr and siAR cells incubated for 24 h with or without CCR2atg. Western blots of STAT3, CCL2 and EMT markers in C4-2 siAR cells incubated for 24 h with or without STAT3 inhibitor (STAT3inh). Western blot of PIAS3 in C4-2 scr and siAR cells. Western blot of PIAS3 in scr and siAR cells of LNCaP ( left ) and LAPC4 ( right ).

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Cell Culture, Neutralization, Migration, Concentration Assay, Control, Proliferation Assay, Incubation, Western Blot

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: IHC (magnification 400× and 100× for inset) staining of CCL2 in 16-week old WT/TRAMP and pesARKO/TRAMP mouse are shown. The breeding strategy to generate WT/TRAMP and MARKO/TRAMP mouse. WT/TRAMP and MARKO/TRAMP mice were confirmed by genotyping. Macroscopic photos ( left ) and haematoxylin eosin (H&E, magnification 40× and 400× for inset, right ) staining of representative metastatic lesions in lung and lymph node of MARKO/TRAMP mouse are shown. Arrows indicate metastatic lesions. Statistical analysis of the number of metastases in WT/TRAMP and MARKO/TRAMP mouse. Graph shows the percentage of mice having metastasis ( n = 9). Fisher's exact test was used. H&E (magnification 100× and 400× for inset) and IHC (magnification is 400×) staining of F4/80 (arrows indicate F4/80 + macrophages), CCL2, pSTAT3, MMP9, and Snail ( left ), and the distribution of staining intensity and statistical analysis ( right ). Chi-square test for trend was used, ( n = 6); bars in graphs, Mean ± SEM.

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: Proliferation assay of TRAMP-C1 scramble (scr) and TRAMP-C1 AR silenced (siAR) cells incubated for 24, 48 and 72 h, ( n = 3); bars in graphs, Mean ± SEM. Western blot of CCL2, STAT3 and AR in TRAMP-C1 scr and siAR cells co-cultured with or without RAW264.7 cells for 24 h. Macroscopic photos of orthotopic tumours in each group, TRAMP-C1 scramble + vehicle (scr veh), TRAMP-C1 siAR + vehicle (siAR veh), and TRAMP-C1 siAR + CCR2 antagonist (siAR CCR2atg) are shown. Quantification of tumour volume in each group (the number of tumours in scr veh, siAR veh and siAR CCR2atg is 18 from 9 mice, 24 from 12 mice, and 33 from 17 mice, respectively). Quantification of TUNEL assay in each group ( left ) and representative pictures ( right ) are shown (magnification is 100×). NC = negative control, ( n = 6); bars , Mean ± SEM in (D and E). Macroscopic photos ( left ) and haematoxylin eosin (H&E, magnification 40× and 400× for inset, right ) staining of representative metastatic lesions in liver and diaphragm of siAR veh mouse are shown. Arrows indicate metastatic lesions. Statistical analysis of the number of metastasis in scr veh, siAR veh and siAR CCR2atg mouse. Graph shows the percentage of the number of mice having metastasis. Fisher's exact test was used.

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Proliferation Assay, Incubation, Western Blot, Cell Culture, TUNEL Assay, Negative Control, Staining

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: H&E (magnification 100× and 400× for inset) and IHC (magnification 400×) staining of F4/80 (arrows indicate F4/80 + macrophages), CCL2, pSTAT3, MMP9, Snail ( left ) and the distribution of staining intensity and statistical analysis ( right ). Chi-square test for trend was used. ( n = 6), bars in graphs, Mean ± SEM.

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: IHC of CCL2 in non-malignant prostate and PCa tissues, representative tissues are shown ( upper panels , magnification 100× and 400× for inset). Fisher's exact test was used for % of positive cases ( lower panel ). IHC of CD68 in non-malignant prostate and PCa tissues, representative tissues are shown ( upper panels , magnification 100× and 400× for inset). Positive cell number in each group are shown ( lower panel ); bars , Mean. Age background of non-malignant and PCa tissue is shown. The serum PSA values between prostate tissues from CCL2-negative and CCL2-positive PCa patients are compared. The number of CD68 positive cells in CCL2-negative and CCL2-positive PCa tissues are shown; bars , Mean ± SEM in (B–E). Overall survival curve of patients with tissue CCL2-negative and CCL2-positive tissues using Kaplan–Meier method is shown. Statistical analysis was done with Log-rank test.

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques:

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: The patient's information from radical prostatectomy is shown. Recurrence-free survival curve of patients with tissue snail-negative/-weakly positive and snail-moderately/-strongly positive tissues using Kaplan–Meier method is shown. Statistical analysis was done with Log-rank test. The association of Snail staining levels and CCL2 staining levels is shown. The association of snail staining levels and pSTAT3 staining levels is shown. Fisher's exact test was used in (C and D).

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques: Staining

Journal: EMBO Molecular Medicine

Article Title: Targeting the androgen receptor with siRNA promotes prostate cancer metastasis through enhanced macrophage recruitment via CCL2/CCR2-induced STAT3 activation

doi: 10.1002/emmm.201202367

Figure Lengend Snippet: The data from this study support the model that AR suppression facilitates metastasis of PCa cells through CCL2/CCR2/STAT3 axis accompanied with increase of macrophage infiltration into PCa site.

Article Snippet: CM collected from monocultures or co-cultures were also used for detection of CCL2 by human CCL2 ELISA kits (R&D Systems) according to the manufacturer's instructions.

Techniques:

Journal: Frontiers in medicine

Article Title: Activated Hepatic Stellate Cells Induce Infiltration and Formation of CD163 + Macrophages via CCL2/CCR2 Pathway.

doi: 10.3389/fmed.2021.627927

Figure Lengend Snippet: FIGURE 3 | The high level of CCL2 in aHSCs was associated with CD163+ macrophage infiltration and increased with liver fibrosis progression. (A) The primary aHSCs are typically fusiform and express the activation marker α-SMA, together with a high expression of CCL2 protein. (B) The aHSCs secrete high levels of CCL2 (Continued)

Article Snippet: When indicated, recombinant human CCL2 protein (rh CCL2, 2ng/ml, R&D Systems, Abingdon, UK) and INCB 3284 (100ng/ml, Tocris Bioscience, UK) were accordingly added, after which the macrophages were harvested, counted, and analyzed.

Techniques: Activation Assay, Marker, Expressing

Journal: Frontiers in medicine

Article Title: Activated Hepatic Stellate Cells Induce Infiltration and Formation of CD163 + Macrophages via CCL2/CCR2 Pathway.

doi: 10.3389/fmed.2021.627927

Figure Lengend Snippet: FIGURE 4 | CCL2 was responsible for macrophages infiltration and differentiation into M2 phenotype during liver fibrosis. (A) Representative images of macrophage infiltration under different chemotaxis treatments including aHSC, aHSC+INCB, Rh CCL2 and medium. (B) Statistical analysis of the number of macrophages (Continued)

Article Snippet: When indicated, recombinant human CCL2 protein (rh CCL2, 2ng/ml, R&D Systems, Abingdon, UK) and INCB 3284 (100ng/ml, Tocris Bioscience, UK) were accordingly added, after which the macrophages were harvested, counted, and analyzed.

Techniques: Chemotaxis Assay