Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Protein array analysis of (A) IMO2B1-conditioned media and (B) control medium. An array was section in order to test unconditioned medium and negative control IMO3A1 at the same time (B). Control medium that was not exposed to IMO cells (top, B) and IMO3A1-conditioned medium that failed to promote neurite outgrowth from chick SAG (batch 11.23; bottom, B) only detected the manufacturer's positive controls (biotinylated HRP conjugate, +) and low levels of gamma interferon (G). All other cytokines were below the level of detection in these media. In contrast, soluble TNF receptor 1 (T), gamma interferon (G), MCP-1 (M), and low levels of RANTES were detected in IMO2B1-conditioned medium shown to promote neurite outgrowth (A, batch 9.13). (− indicates negative controls, + positive controls).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Protein Array, Negative Control

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Immunofluorescence (green) representing the secondary antibody binding to the hair cell marker myosin VI was detected in hair cells of dissociated E14 mouse inner ear cultures. The dissociated cells form aggregates of hair cells and supporting cells. MCP-1 was also prominently expressed in hair cells (red), as well as in some unidentified cells surrounding the aggregate. Supporting cells within the aggregate were negative for MCP-1. The third panel demonstrates the areas where myosin VI and MCP-1 immunofluorescence overlaps (yellow). These results demonstrate that hair cells express MCP-1. Magnification, ×630.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Immunofluorescence, Binding Assay, Marker

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Western blot analysis demonstrated MCP-1-like protein in active IMO2B1-conditioned medium when concentrated by filtration column chromatography. Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated). Molecular weight standards (kDa) are indicated at the left.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Western Blot, Filtration, Column Chromatography, Recombinant, Molecular Weight

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: E5 chick SAG treated with IMO2B1 produced neurite outgrowth (mean 2.6, SE 0.6, n = 5,batch 9.22). Addition of 150 ng/ml of anti-MCP-1 antibody to the IMO2B1 medium decreased outgrowth of SAG significantly (mean 0.6, SE 0.3, n = 5, p < 0.01, batch 9.22).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Produced

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: (A) IMO2B1-conditioned medium promoted neurite outgrowth of E5 chick SAG (batch 9.22; explant score = 4). (B) Addition of anti-MCP-1 antibody (150 ng/ml) decreased E5 chick SAG outgrowth (explant score = 1.5). The photo shows the area of maximal outgrowth. Magnification, ×100 (A), ×200 (B).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques:

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: Monocyte migration was measured using a classic chemotactic assay. The number of migrating cells is depicted minus the background number of cells migrating in control conditions (mean 58 cells, SE 19, n = 7). In the presence of MCP-1 (20 ng/ml), monocytes migrated to the side of the chamber containing the protein (mean 299, SE, 40, n = 4). Addition of the function-blocking anti-MCP-1 antibody reduced the migration of the monocytes significantly (mean 46, SE 31, n = 3, p < 0.01). IMO2B1 alone also induced the migration of monocytes above background levels (mean 58, SE 14, n = 7, batch 2.05) and the function-blocking antibody decreased this migration significantly (mean 11, SE 8, n = 6, p < 0.01).

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Migration, Chemotaxis Assay, Blocking Assay

Journal:

Article Title: Immortalized Mouse Inner Ear Cell Lines Demonstrate a Role for Chemokines in Promoting the Growth of Developing Statoacoustic Ganglion Neurons

doi: 10.1007/s10162-005-0013-8

Figure Lengend Snippet: IMO2B1-conditioned medium produced outgrowth from E5 chick SAG (IMO2B1 batch 9.13, mean 3.4, SE 0.35, n = 7). When anti-MCP-1 function-blocking antibody was added at a concentration of 10 or 25 ng/ml (n = 14 total) the amount of outgrowth was reduced significantly (mean 1.97, SE 0.42, n = 14, p < 0.01). Supplementing the anti-MCP-1-treated IMO2B1-conditioned medium with MCP-1 (“add back,” 20 ng/ml) led to a statistically significant increase in SAG neurite outgrowth (mean 3.72, SE 0.31, n = 11, p < 0.01) above that observed with anti-MCP-1 only, indicating a direct role for MCP-1 in promoting SAG outgrowth. No further increase was noted when 200 ng/ml of MCP-1 (mean 2.7, SE 0.42, n = 5; p > 0.01) was added to the antibody-treated cultures.

Article Snippet: Lane 1: recombinant mouse MCP-1 (100 ng/ml; R&D Systems); lane 2: IMO2B1-conditioned medium of (batch 4.22); lane 3: IMO2B1-conditioned medium (batch 4.25); lane 4: IMO2B1 (batch 4.28); lane 5: IMO2B1-conditioned medium (batch 5.11); lane 6: recombinant mouse MCP-1 (200 ng/ml, R&D Systems); lane 7: IMO2B1-conditioned medium (batch 4.22 concentrated); lane 8: IMO2B1-conditioned medium (batch 4.25, concentrated); lane 9: IMO2B1-conditioned medium (batch 4.28 concentrated); lane 10: IMO2B1-conditioned medium (batch 5.11 concentrated).

Techniques: Produced, Blocking Assay, Concentration Assay

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (A) or CCL2 (B) at concentrations of 5, 10, 50, or 100 ng/mL, and cell viability was determined by cell counting using a hemocytometer or MTT assay, respectively. (C) Flow cytometry results are presented as histograms of the average percentage of cells that expressed IGF-1R and CCR2 receptors (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 4/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to the control group: p < 0.05 (*) or p < 0.0001(***); significant value compared to control group and the other treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Cell Counting, MTT Assay, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by fluorescence microscopy. ( A ) Photomicrographs show the expression of FN ascertained by immunofluorescence and fluorescence microscopy analysis. Magnification: 400× ( B ) Bars correspond to the quantitative analysis of FN expression in tEnd.1 cells in selected microscopic fields (n = 5/group). The results are expressed in pixels/μm 2 . ( C ) Flow cytometry results are presented as histograms of the average percentage of cells that expressed CD49e/VLA-5 and CD44 receptors for FN (gray) and immunoglobulin control. Values and bars are represented as the mean ± SEM (n = 5/group). Results were analyzed by one-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.0001 (***); significant values compared to control group and single treatments: p < 0.0001 (#).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Fluorescence, Microscopy, Expressing, Immunofluorescence, Flow Cytometry, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: (A) tEnd.1 cells treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h on BSA or FN coating were stained with Alexa 488-phalloidin and analyzed by confocal microscopy with a 63× objective. ( B ) tEnd.1 cells were allowed to adhere on BSA- or FN-coated surfaces for 1 h after stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 24 h. ( C ) tEnd.1 cells were allowed to migrate through transwell chambers coated with BSA or FN after chemotactic stimulation with IGF-1, CCL2, or IGF-1/CCL2 for 6 h. Photomicrographs demonstrate cells invading through the transwell membrane. Giemsa staining. Scale bar = 10 μm. ( D ) Bars represent the number of migrating cells in a transwell system. Data are represented as mean ± SEM (n = 5/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant values compared to control group: p < 0.05 (*), p < 0.01 (**), or p < 0.0001 (***); significant values compared to control group and the IGF-1 treatment: p < 0.05 (#); and significant values compared to control group and single treatments: p < 0.01 (+).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Staining, Confocal Microscopy, Membrane, Control

Journal: PLoS ONE

Article Title: Combined Effect of Insulin-Like Growth Factor-1 and CC Chemokine Ligand 2 on Angiogenic Events in Endothelial Cells

doi: 10.1371/journal.pone.0121249

Figure Lengend Snippet: ( A ) tEnd.1 cells were treated with IGF-1 (100 ng/mL), CCL2 (10 ng/mL), or a combination of both for 24 h and analyzed by optical microscopy. Photomicrographs show intracellular lumina in tEnd.1 cells, indicated by arrows. Giemsa staining. Scale bar = 10 μm. ( B ) tEnd.1 cells were treated with IGF-1, CCL2, or IGF-1/CCL2 for 8 days on BSA or FN coating and analyzed by optical microscopy. Photomicrographs demonstrate capillary-like structures, indicated by asterisks. Giemsa staining. Scale bar = 10 μm. ( C ) Number of capillary-like structures. ( D ) Luminal area of capillary-like structures. Bars represent the mean ± SEM (n = 6/group). Results were analyzed by two-way ANOVA followed by Bonferroni’s post-test. Significant compared with control, p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).

Article Snippet: Cells were then treated with recombinant mouse CCL2/JE/MCP-1 (CCL2) (R&D Systems, Minneapolis, MN, USA) at concentrations of 5, 10, 50, and 100 ng/mL for 24 h. After treatment, cells were incubated with 5 mg/mL of tetrazolium salt (MTT) (Sigma-Aldrich) diluted in RPMI 1640 with 2% FBS.

Techniques: Microscopy, Staining, Control

Journal: Frontiers in Immunology

Article Title: CCR2 + Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

doi: 10.3389/fimmu.2022.835986

Figure Lengend Snippet: CCR2/CCL2 axis promoted pro-inflammatory macrophages through the phosphorylation of p65. (A) Relative mRNA expression of Nos2 in untreated, CCL2 treated and LPS treated macrophages. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (B) Representative immunofluorescence staining of iNOS in untreated, CCL2 treated and LPS treated macrophages. (C) Relative mRNA expression of inflammatory factors in macrophages treated with LPS or IL-4, and RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (D) Relative mRNA expression of Ccr2 in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. (E, F) Relative mRNA expression of pro-inflammatory (E) and anti-inflammatory factors (F) in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. ns, no significance. (G) Representative immunofluorescence staining of p65 in macrophages in untreated, treated with CCL2, and CCL2+RS504393. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05. **P <0.01. (H) Relative mRNA expression of inflammatory factors Il6 , Il1β and nos2 treated with CCL2 or p65 inhibitor. Data is from 3 independent experiments. Values are mean ± SD. *P < 0.05, ns, no significance.

Article Snippet: BMDMs were pretreated with P65 inhibitor Helenalin (10μm, HY-119970; MCE) for 2 hours, then treated with CCL2 (100ng/ml, 479-JE; R&D) for 3 hours.

Techniques: Expressing, Immunofluorescence, Staining

Journal: Frontiers in Immunology

Article Title: CCR2 + Macrophages Promote Orthodontic Tooth Movement and Alveolar Bone Remodeling

doi: 10.3389/fimmu.2022.835986

Figure Lengend Snippet: CCL2 treatment promoted the phosphorylation of p65 in macrophages. (A) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 for 0, 30, 60 and 360 minutes. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (B) Western blot analysis of p-p65 and p65 expression in macrophages treated with CCL2 and RS504393. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. ns, no significance. (C, D) Western bolt analysis of p-p65 and p65 expression in macrophages treated with CCL2 or not, and with Ccr2 siRNA. Data is from 3 independent experiments. Densitometric quantitation with Image J. Values are mean ± SD. *P < 0.05. (E, F) The distance of OTM in TM and TM+CCR2i group and representative 3D micro-CT reconstruction. White arrow: direction of force and tooth movement. Values are mean ± SD. n = 5. ***P < 0.001. (G) Graphic abstract of this study.

Article Snippet: BMDMs were pretreated with P65 inhibitor Helenalin (10μm, HY-119970; MCE) for 2 hours, then treated with CCL2 (100ng/ml, 479-JE; R&D) for 3 hours.

Techniques: Western Blot, Expressing, Quantitation Assay, Micro-CT