human ccl11 eotaxin kit  (R&D Systems)

Journal: bioRxiv

Article Title: Sympathetic Nervous System Overactivation Induces Colonic Eosinophil-Associated Microinflammation and Contributes to the Pathogenesis of Irritable Bowel Syndrome

doi: 10.1101/2024.07.22.604223

Figure Lengend Snippet: (A) PCR analysis of the expression of eosinophil recruitment genes ( Ccl11, Cccl24, Ccl26, Il5, and Il33 ) in the colons of vehicle (Veh)- and NE-treated rats 6 h after NE treatment. (B) Bar graphs showing relative changes in gene expression between the colons of Veh- and NE-treated rats (n = 5 in each group). (C) Summary data of the number of eotaxin-1-ir cells at different time points after NE treatment. *P < 0.05, vs. vehicle (Veh). (D) Immunohistochemistry images showing eotaxin-1 expression in the colon 6 h after NE treatment. Scale bar, 50 μm. (E) Immunohistochemistry images showing co-expression of eotaxin-1 with vimentin, CD68, and CD31 in the colon of Veh- and NE-treated rats, respectively. Enlarged images of the white dotted frame are shown at the bottom. Black arrows indicate double-labelled cells, and white arrows indicate single-labelled cells. Scale bar, 20 μm. (F) Bar graphs showing the eotaxin-1-ir cells in the Veh and NE stimulation groups (n =7 in each group). * P < 0.05, eotaxin-1-ir cells NE vs Veh. (G) Bar graphs showing data of eotaxin-1 cells, CD68 cells, and vimentin cells in the Veh- and NE-treated rats (n =7 in each group). (H) Bar graphs showing the number of eotaxin-1-ir cells with or without phentolamine (Phe) and propranolol (Pro) administration in Veh- and NE-treated rats (n= 7 in each group). (I) Bar graphs showing the number of MBP-ir cells with or without SB328437 administration in Veh- and NE-treated rats (n= 6 rats in each group). Data are presented as mean ± SEM. A one-way ANOVA was used for the analysis shown in (C, H), a two-way ANOVA with Bonferroni post hoc test was used for the analysis shown in (I), and an unpaired t -test was used for the analysis shown in (B, F, G). *P < 0.05, **P < 0.01.

Article Snippet: Eotaxin-1 release from CCD-18co cell lines following NE stimulation (0, 1, and 10 μM NE after 1, 3, or 6 h of incubation) was measured using the human CCL11/Eotaxin kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s protocol.

Techniques: Expressing, Immunohistochemistry

Journal: Frontiers in Immunology

Article Title: Eotaxin-1/CCL11 promotes cellular senescence in human-derived fibroblasts through pro-oxidant and pro-inflammatory pathways

doi: 10.3389/fimmu.2023.1243537

Figure Lengend Snippet: CCL11 induces positive feedback on CCL11 secretion and increases SASP mediators. MRC-5 cells were pre-treated with rhCCL11 (500ng/10 5 cells/mL) for 24 hours and supernatant was replaced for fresh media without rhCCL11 for additional 24 hours for cytokine secretion quantification. (A) Increased CCL11 secretion on cells pre-treated with rhCCL11 evaluated by ELISA. (B–G) Increased secretion of the SASP cytokines IL-6 and IL-8 after 24 hours treatment, with no differences observed in the remained cytokines (N = 6). (A) Mann-Whitney U-test for non-parametric samples was performed. (B–G) Unpaired t-test for normal distribution was performed. Data are presented as median and IQR. Significant differences considered when P<0.001 (***) or P<0.0001 (****).

Article Snippet: Eotaxin-1/CCL11 levels were measured by enzyme-linked immunosorbent assay (ELISA) (Human Eotaxin (CCL11) Standard ABTS ELISA Development Kit, Peprotech) according to the manufacturer’s instructions from 100μL of conditioned media stored at -80° C.

Techniques: Enzyme-linked Immunosorbent Assay, MANN-WHITNEY

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