Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Characterization of CAFs and NFs obtained from clinical surgical tissues from patients with head and neck cancer (HNC) ( a ) Morphological comparisons between CAFs and NFs from a representative HNC case showed that CAFs (right panel) consisted of more cytoplasmic protrusions than NFs (left panel). Photographs were captured at 40× magnification. ( b ) Quantitative PCR (left panel) of the culture medium showed a significantly higher expression of vimentin and α-SMA in CAFs than in NFs. Western blot analysis (right panel) also demonstrated that levels of vimentin and α-SMA were significantly higher in CAFs than in NFs. ( c ) Flow cytometric analysis of the cell surface markers, CD10 and GPR77, showed a marked increase in their expression in CAFs compared to NFs. ( d ) A heat map of the gene microarray of NFs and CAFs showed that there were several differences in the expression profile of the secreted genes. The arrow indicates a marked discrepancy of the relative mRNA levels of CCL11 in CAF compared with NF. ( e ) The RT-PCR (left panel) and ELISA (right panel) analysis showed an increased expression of CCL11 in CAFs compared with that in NFs. ( f ) Western blot analysis showed that the protein level of CCL11 was higher in CAFs than in NFs in cell lysates. ( g ) Western blot analysis showed a higher CCL11 expression in CAF-CM compared to NF-CM. The asterisk indicated a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Western Blot, Microarray, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 produced by CAFs causes increased migration and invasion, and the EMT of HNC cells. ( a ) Comparative analysis of the migration and invasion of HNC cells associated with CCL11. Four test groups were classified for comparative analysis of migration and invasive abilities. FaDu and NPC204 cells cultured with medium containing CAF-induced CCL11 presented greater abilities of migration and invasion, with a statistically significant difference, than three other groups: NF, NF with CCL11, and CAFs treated with CCL11 antibody. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) ( b ) Comparative photographs of the infiltrating behavior of FaDu and NPC204 cells in an organotypic culture in four groups seeded onto a mixture layer containing NFs or CAFs with CCL11 or CCL11 antibody. The arrow(s) indicate infiltration buds from the HNC cells seeded above. ( c ) Representative blots of the EMT-associated markers in FaDu and NPC204 cells, as observed upon Western blotting analysis in five groups, showed that treatment with CAF-conditioned medium or the application of rCCL11 decreased the expression of epithelial-type markers (E-cadherin), and increased the expression of mesenchymal-type markers (fibronectin) and EMT regulators (Snail and Twist). In addition, increased expression of invasion-related MMP2 and MMP9 was also seen in those two groups, compared with other groups. The asterisk indicates a significant difference ( p < 0.05) between experimental and control groups. Results are expressed as mean ± SD.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Produced, Migration, Cell Culture, Western Blot, Expressing, Control

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: Comparative analysis of induction of CSC properties and drug resistance in HNC cells associated with CCL11. ( a ) Four groups were classified for comparative analysis of the ability of sphere formation. Increased ability of sphere formation in two test groups of HNC cells exposed to a CAF medium and the group with treatment of rCCL11 was noted. ( b ) Flow cytometric analysis showed a significant increase in CD44 and CD44/CD24, as well as in CD133 in HNC cells exposed to rCCL11, compared to control HNC cells ( p < 0.05). ( c ) Flow cytometric analysis showed a marked increase in ALDH-1 activity in HNC cells exposed to rCCL11 compared to control HNC cells. ( d ) Western blot analysis showed that CSC-representative markers, Oct-4, Nanog, and Sox-2, were also overexpressed in addition to the increased expression of two important drug resistance genes, ABCG-2 and MDR-1 , in HNC cells exposed to rCCL11. ( e ) Treatment with Cisplatin at 24 h showed a significant increase in chemoresistance in both FaDu and NPC204 cells exposed to rCCL11 compared with control HNC cells. The asterisk indicates a significant difference (*: p < 0.05; **: p < 0.01) between experimental and control groups.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Control, Activity Assay, Western Blot, Expressing

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: CCL11 and CCR3 expression with associated signal pathway in HNC cell lines and their correlation to clinical outcomes in 104 HNC patients. ( a ) Confocal microscopic images showed CCL11 (green) localized to both cell and nuclear membranes, while CCR3 (red) localized only to the cell membrane in FaDu cells; CCL11 and CCR3 co-localized at the cell membrane (yellow). In NPC204 cells, CCL11 (green) and CCR3 (red) were found to co-localize at protrusions polarized to the cells (yellow). ( b ) Using the crisp technique, higher expression of CCR3, MMP2, and MMP3 was found in over-expressed CCL11 cloned-FaDu and NPC204 cells. Cloned CCL11-overexpressed cells were abolished by adding eotaxin siRNA or CCR3 antibody, which reversed the expression of CCR3 and invasion-related MMP2 and MMP9. ( c ) Higher phosphorylation levels of p38 MAPK and ERK were found in cloned CCL11-overexpressed FaDu and NPC204 cells and were reversed by treatment of the p38 MAPK inhibitor (SB203580) and ERK inhibitor (FR180204), respectively. The phosphorylation level of JNK was kept in low condition before and after treatment of the JNK inhibitor (SP600125). ( d ) Photomicrographs of immunohistochemical staining from tissue microarray showing CCL11 and CCR3 expression in three different representative groups of HNC patients (magnification, ×200). ( e ) Kaplan–Meier survival analysis of patients showed that overexpression of CCL11 and CCR3 were statistically associated with poor overall survival).

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Expressing, Membrane, Clone Assay, Phospho-proteomics, Immunohistochemical staining, Staining, Microarray, Over Expression

Journal: Cancers

Article Title: Cancer-Associated Fibroblasts Promote Tumor Aggressiveness in Head and Neck Cancer through Chemokine Ligand 11 and C-C Motif Chemokine Receptor 3 Signaling Circuit

doi: 10.3390/cancers14133141

Figure Lengend Snippet: The diagrammatic illustration demonstrates the major mechanism that CAFs secreting CCL11 promotes HNC cell migration and invasion and induces properties of drug resistance and stemness, shown as follows. CAFs in TME secret CCL11 binding to the CCR3 receptors on HNC cells via the paracrine effect. The signal induces overexpression of transcriptional factors, such as Snail and Twist, which regulate EMT and are also responsible for self-induction of CCL11 in an autocrine fashion. As a result, CCL11, via paracrine or autocrine signaling when targeting CCR3 receptors, play a functional role in the induction of EMT and CSC properties, for further tumor progression.

Article Snippet: CAFs and NFs supernatant CCL11concentrations were measured using the CCL11 (human) ELISA kit (R&D system) following the manufacturer’s protocol.

Techniques: Migration, Binding Assay, Over Expression, Functional Assay

Journal: Molecular medicine reports

Article Title: Analysis of murine and human Treg subsets in inflammatory bowel disease.

doi: 10.3892/mmr.2017.6912

Figure Lengend Snippet: Figure 5. Serum inflammatory cytokines in patients with ulcerative colitis. Serum from ulcerative colitis patients and healthy donors were collected and inflammatory cytokines were determined by ELISA. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001 as indicated. IL, interleukin; TSLP, thymic stromal lymphopoietin; MMP, matrix metalloproteinase.

Article Snippet: Serum cytokine levels were analyzed using the following commercially available ELISA kits according to the manufacturer's protocol: MMP-2, Total MMP-2 Quantikine ELISA kit (cat. no. MMP200; Quantikine, R&D Systems, Inc., Minneapolis, MD, USA); thymic stromal lymphopoietin, Legend MaxTM Human TSLP ELISA kit with Pre-coated Plates (cat. no. 434208; BioLegend, Inc., San Diego, CA, USA); MMP-9, Legend MaxTM Human MMP-9 ELISA kit with Pre-coated Plates (cat. no. 440707; BioLegend, Inc.); IL-17A, Legend MaxTM Human IL-17A ELISA kit with Pre-coated Plates (cat. no. 433918; BioLegend, Inc.); IL-25, Human IL-17E/IL-25 AccuSignal ELISA kit (cat. no. KOA0468; Rockland Immunochemicals, Inc., Limerick, PA, USA); and eotaxin, Human Eotaxin ELISA kit (cat. no. KOA0159; Rockland Immunochemicals, Inc.).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Cell reports

Article Title: Innate Lymphoid Cells Play a Pathogenic Role in Pericarditis

doi: 10.1016/j.celrep.2020.02.040

Figure Lengend Snippet: (A) Expression of the Ccl11 gene encoding eotaxin-1 in heart homogenates was analyzed by qPCR. (B) Schematic description of cardiac fibroblasts co-culture with ILC2s separated by 0.4-μm transwell. (C) Expression of Ccl11 in cardiac fibroblasts was analyzed by qPCR. (D) Eotaxin-1 concentrations in cell culture supernatants were measured by ELISA. IL-2 and IL-7 were included in culture media and IL-33 was added where indicated (C and D). (E–H) Expression of genes in heart homogenates from WT, Rag2 −/− , and Rag2 −/− Il2rg −/− mice treated with IL-33 was analyzed by qPCR. Ccl11 (E), Ccl24 (F), Il5 (G), and Il13 (H). (I) Immunohistochemistry staining for CCL11 of heart sections from WT mice treated with PBS or IL-33. Bars: 50 μm. Data are representative of two independent experiments and displayed as the mean with SD. Unpaired t test (A) or one-way ANOVA followed by Tukey’s post hoc test (C–H) was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001. See also .

Article Snippet: After deparaffinization, heat-induced antigen retrieval and blocking, sections were stained with 5 μg/ml Polyclonal Goat Anti-Mouse CCL11 antibody (R&D Systems) and Anti-Goat HRP-DAB Cell & Tissue Staining Kit (R&D Systems) and counter stained with Hematoxylin.

Techniques: Expressing, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining

Journal: Cell reports

Article Title: Innate Lymphoid Cells Play a Pathogenic Role in Pericarditis

doi: 10.1016/j.celrep.2020.02.040

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: After deparaffinization, heat-induced antigen retrieval and blocking, sections were stained with 5 μg/ml Polyclonal Goat Anti-Mouse CCL11 antibody (R&D Systems) and Anti-Goat HRP-DAB Cell & Tissue Staining Kit (R&D Systems) and counter stained with Hematoxylin.

Techniques: Recombinant, Staining, cDNA Synthesis, SYBR Green Assay, Blocking Assay, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Software

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL11 (Eotaxin) Induces CCR3-Dependent Smooth Muscle Cell Migration

doi: 10.1161/01.atv.0000131654.90788.f5

Figure Lengend Snippet: Figure 3. CCL11 induces mouse SMC chemotaxis. A, Mouse CCL11 in 0.3% FCS at the concentrations indicated (ng/mL) were placed in the bottom wells of a modified Boyden Cham- ber. 104 SMCs were placed in the top wells. The chamber was incubated for 6 hours at 37°C and then analyzed for migration as described in Materials and Methods. For comparison, recom- binant PDGF-BB (1 ng/mL) or 10% FBS were also used as che- moattractants. 0.3% serum was used as negative control. *P0.005 compared with 0 CCL11. B, Checkerboard analysis in which CCL11 (C; 100 ng/mL) was placed in the bottom, top, or both wells as indicated. *P0.005 compared with 0.3% FCS (–). C, SMC were incubated with antibodies against CCR3 (anti- CCR3), CCR2 (anti-CCR2) (1 g/mL), or irrelevant IgG for 30 minutes at 37°C before being placed in the Boyden chamber. Results are expressed as SMC/hpf SEM and represent tripli- cate experiments *P0.005 compared with 0.3% FCS. #P0.05 compared with 0.3% FCS.

Article Snippet: Recombinant mouse CCL11 (420-ME; R & D Systems, Minneapolis, MN) was added to the bottom chamber in 0.3% FBS and DMEM.

Techniques: Chemotaxis Assay, Incubation, Migration, Comparison, Negative Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL11 (Eotaxin) Induces CCR3-Dependent Smooth Muscle Cell Migration

doi: 10.1161/01.atv.0000131654.90788.f5

Figure Lengend Snippet: Figure 5. CCL11 does not modulate SMC proliferation. SMCs were plated in 35-mm wells and treated with CCL11 (10 ng/mL or 100 ng.ml) PDGF (1 ng/mL), CCL11 (100 ng/mL) PDGF (1 ng/mL), 10% FCS, or 0.3% FCS. Duplicate plates were counted on the days indicated. The medium, with fresh growth factors, was replaced every 48 hours. Results are expressed as cell number SEM and represent the average of duplicate experiments. Figure 4. CCL11 induces mouse SMC migration. SMCs were grown to confluence and then incubated in DMEM 0.1% BSA for 24 hours. Linear wounds were made in each plate and SMCs were then incubated with DMEM 0.1% BSA alone (con- trol) or in the presence of CCL11 (concentration in ng/mL indi- cated on the X axis). In some experiments, SMCs were preincu- bated for 1 hour with anti-CCR3 (CCR3 Ab) or an irrelevant antibody (IgG) before wounding and treatment with 10 ng/mL CCRL11. The wound area was measured immediately after wounding and at 24 hours. Migration is expressed as the % decrease in wound area in 24 hours compared with control. Experiments were done in duplicate, with 3 to 5 plates used for each condition. *P0.001, 10 ng/mL CCL11 compared with control; **P0.001, 10 ng/mL eotaxin CCR3 Ab compared with 10 ng/mL eotaxin alone; #NS, 10 ng/mL eotaxin IgG compared with 10 ng/mL eotaxin alone.

Article Snippet: Recombinant mouse CCL11 (420-ME; R & D Systems, Minneapolis, MN) was added to the bottom chamber in 0.3% FBS and DMEM.

Techniques: Migration, Incubation, Concentration Assay, Control

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CCL11 (Eotaxin) Induces CCR3-Dependent Smooth Muscle Cell Migration

doi: 10.1161/01.atv.0000131654.90788.f5

Figure Lengend Snippet: Figure 6. Expression of CCR3 antigen in injured mouse femoral arteries. Samples were obtained from a previous study in which C57Bl/6 mice had undergone wire-induced femoral arterial injury.39 Sections from uninjured arteries (A, B), arteries harvested 5 days (C, D), 7 days (E, F), and 28 days after injury (G through J) were stained with antibodies to CCR 3 (left) or CCL11 (right). Magnifica- tion40 for A, B, E, F, G, and H and 200 for C, D, I, and J. Sections are representative of studies done on 3 different animals.

Article Snippet: Recombinant mouse CCL11 (420-ME; R & D Systems, Minneapolis, MN) was added to the bottom chamber in 0.3% FBS and DMEM.

Techniques: Expressing, Staining