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:

Article Title: Protection against inflammation- and autoantibody-caused fetal loss by the chemokine decoy receptor D6

doi: 10.1073/pnas.0607514104

Figure Lengend Snippet: Chemokines in the LPS model of fetal loss. (A–E) Serum chemokine concentrations after LPS treatment in WT and D6−/− male mice. WT (open symbols) and D6−/− (filled symbols) mice were injected i.p. with 1.35 mg/kg LPS. At the indicated time points, circulating chemokine concentrations were measured by ELISA. Data are from seven mice for each time point. (F–J) Serum chemokine concentrations. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Circulating chemokine concentrations were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. (K–O) Chemokine levels in placenta. WT (open columns) and D6−/− (filled columns) mice at day 10 of pregnancy were injected i.p. with 0.4 mg/kg LPS. Chemokine concentrations (expressed as nanograms of chemokine per milligram of total proteins of the lysates) were measured at 8 h postinjection by ELISA. Data are from nine WT and eight D6−/− mice. Results are reported as mean ± SEM. (A, F, and K) CCL22. (B, G, and L) CCL2. (C, H, and M) CCL11. (D, I, and N) CCL3. (E, J, and O) CXCL2.

Article Snippet: To block inflammatory chemokines, animals were treated with a mixture of goat antibodies to the mouse CC chemokines CCL3L1 (catalog no. AB450NA), CCL4 (catalog no. AB451NA), CCL2 (catalog no. AB479NA) and rat mAb anti-mouse CCL5 (catalog no. MAB478) purchased lyophilized from R&D Systems, resuspended in PBS, and mixed.

Techniques: Injection, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 1. Effects of epigallocatechin-3-gallate (EGCG) on oncostatin M (OSM)–stimulated CCL2 production and MEK/ERK phosphorylation. A, Human bone marrow–derived osteoblasts were incubated for various periods of time with 10 ng/ml OSM, and the CCL2 mRNA levels were assayed by Northern blotting. B and C, Human bone marrow–derived osteoblasts (B) and MG-63 cells (C) were incubated for 8 hours with OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM), and CCL2 mRNA levels were subjected to Northern blotting. D and E, Cells were incubated for 24, 48, or 72 hours with OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM). CCL2 released into the culture media of human bone marrow–derived osteoblasts (D) and MG-63 (E) was quantified by enzyme-linked immunosorbent assay. Open bars indicate OSM; solid bars indicate OSM combined with EGCG. Values are the mean and SD results from 3 independent experiments. P 0.05 versus OSM at 0 hour; P 0.05. F, MG-63 cells were incubated for 20 minutes with OSM, alone or in combination with EGCG, and were assessed for phosphorylation at MEK-1/2 (Ser217/221) and ERK-1/2 (Thr202/Tyr204).

Article Snippet: Anti-human CCL2 antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Phospho-proteomics, Derivative Assay, Incubation, Northern Blot, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 4. Effects of EGCG on OSM-induced activator protein 1 (AP-1)–DNA binding and AP-1–CCL2 promoter interaction in MG-63 cells. A–C, Cells were incubated for 30 minutes with 10 ng/ml OSM, alone or in combination with 10 g/ml EGCG (3 hours before the addition of OSM). The expression of c-Fos (A) and c-Jun (B) mRNA was determined by Northern blot analysis, and the respective protein levels were examined by Western blotting (C). D, Nuclear extracts from cells were subjected to electrophoretic mobility shift assay with supershift, using anti–c-Fos antibody. E, Cells were also subjected to chromatin immunoprecipitation (IP) assay with the indicated antibodies or with normal rabbit serum as control (mock). Immunoprecipitates from each sample, including input and mock, were analyzed by polymerase chain reaction using primers specific for the AP-1 binding region of human CCL2 promoter. Ac-H3 acetylated histone 3 (see Figure 1 for other definitions).

Article Snippet: Anti-human CCL2 antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Binding Assay, Incubation, Expressing, Northern Blot, Western Blot, Electrophoretic Mobility Shift Assay, Chromatin Immunoprecipitation, Control, Polymerase Chain Reaction

Journal: Arthritis and rheumatism

Article Title: Epigallocatechin-3-gallate diminishes CCL2 expression in human osteoblastic cells via up-regulation of phosphatidylinositol 3-Kinase/Akt/Raf-1 interaction: a potential therapeutic benefit for arthritis.

doi: 10.1002/art.23937

Figure Lengend Snippet: Figure 6. Immunolocalization of CCL2 and CD68 in the ankle joints of rats with collagen-induced arthritis. A–D, Sections from the control group. A, Pannus (P) formation and obliteration of joint space (JS). B, High-power view of A, showing erosion of cartilage and bone by pannus. C, Marked expression of CCL2 in osteoblasts (arrowheads) overlying osteolytic areas and mononuclear round cells (arrows). D, Clearly visible CD68 in macrophages (arrowheads) and osteoclasts (arrows) adjacent to resorption lacunae. E–H, Sections from the group treated with epigallocatechin- 3-gallate. E, Preservation of joint space, cartilage, and bone. F, High-power view of E, showing minimal inflammatory cell infiltration and cartilage erosion. G, Diminished numbers of CCL2 osteoblasts (arrowheads). H, Decreased infiltration of CD68 macrophages (arrowheads) and osteoclasts (arrows). (Hematoxylin and eosin staining in A, B, E, and F; avidinbiotinperoxidase staining in C, D, G, and H. (Original magnification 25 in A and E; 200 in B, C, D, F, G, and H.)

Article Snippet: Anti-human CCL2 antibody was obtained from R&D Systems (Minneapolis, MN).

Techniques: Control, Expressing, Preserving, Staining