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: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 3. The accumulation of cells 24 h after CCL3 injection is hampered in NOD (5–7-week-old) mice (A). Granulocytes (B) and monocytes (C) are not attracted in NOD mice. In response to CCL2, NOD mice show a deficient accumulation of cells into the air pouch after 24 h (D). The accumulation of monocytes to CCL2 is almost absent in NOD (E). Data are cumulative of two individual experiments and represented as mean SEM (n=6/group in A and C; n=4/group in B, C, E). *p<0.05 relative to NOD as determined with Mann-Whitney (A, D) or Student's t-test (B, C, E).

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Injection, MANN-WHITNEY

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 6. The air pouch of C57BL/6 and BALB/c contains a high number of MU (A) and DC (B) after CCL2 injection, while those of NOD mice do not. MU and DC are defined as shown in Fig. 4. Data are cumulative of two experiments and are represented as mean SEM; each bar represents at least four animals (5–7 weeks old), *p<0.05 as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Injection

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 7. The air pouch of control mice at 24 h after CCL2 injection shows an increased production of IL-1b; however, this is not seen in NOD (A). NOD mice produce higher amounts of IL-10 in comparison to C57BL/6 and BALB/c mice (B). A representative flow cytometric analysis of intracellular staining for IL-10 (gated for CD11b+ cells) shows that monocytes (CD11bhiF4/80medCD11clow) are producing IL-10, while MU (CD11b+F4/80hiCD11clow) do not (C). Figures shown are representative of five NOD mice 24 h after injection with CCL2. The production of TNF-a in the air pouch of NOD mice is similar to control mice (D). Data are represented as mean SEM (n=5/group; 5–7 weeks old). *p<0.05 relative to NOD as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Control, Injection, Comparison, Staining

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 9. Peritoneal exudate MU of NOD mice show a decreased migration towards CCL2 compared to C57BL/6 control mice using a Boyden chamber chemotaxis assay. Data are represent mean SD (n=3), *p<0.05 relative to NOD as determined with Student's t-test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Migration, Control, Chemotaxis Assay

Journal: European journal of immunology

Article Title: NOD mice have a severely impaired ability to recruit leukocytes into sites of inflammation.

doi: 10.1002/eji.200425513

Figure Lengend Snippet: Fig. 8. Neutralization of IL-10 does not restore the cell recruitment towards CCL2 to the level of C57BL/6 mice (A). Addition of recombinant IL-10 to the air pouch of C57BL/6 mice decreases the recruitment towards the control level (A). Blocking IL-10 raises the level of IL-1b in the air pouch of NOD to a similar level of C57BL/6 mice (B). Addition of recombinant IL-10 in the air pouch of C57BL/6 does not lower the amount of IL-1b (B). Mean SD (n=6) are shown. Data in (B) are presented relative to the amount of IL-1b that is observed in control-injected animals. Statistical significance was deter- mined with Kruskall-Wallis test and differences between groups using the Mann-Whitney test.

Article Snippet: To study the effect of IL-10, the air pouchwas injected either with 1 ml CCL2 in combination with an mAb against IL-10 (0.5 mg/ml; JES-2A5.1, produced in our own laboratory) or with CCL2 in combination with recombinant mouse IL-10 (0.25 lg/ml; R&D systems).

Techniques: Neutralization, Recombinant, Control, Blocking Assay, Injection, MANN-WHITNEY

Journal: Neuro-oncology Advances

Article Title: Immune deconvolution and temporal mapping identifies stromal targets and developmental intervals for abrogating murine low-grade optic glioma formation

doi: 10.1093/noajnl/vdab194

Figure Lengend Snippet: CD8 + T cell predominance is governed by NF1/RAS regulation of Ccr4 expression. A, CD8 + T cells content in Nf1 -OPG optic nerves ( n = 10) is similar to controls ( n = 8) at 3, 4.5, 6, and 9 WOA, but increases by 12 WOA ( P < 0.0001). Nf1 +/– mice ( n = 6) have similar numbers of CD8 + T cells as controls from 3 to 12 WOA. B, CD4 + T cell content does not change in Nf1 -OPG optic nerves relative to controls at 3, 4.5, 6, 9, and 12 WOA. C, Ccl2 did not increase CD4 + T cell migration in WT or Nf1 +/– mice. Ccl12 or medium (control) does not increase CD4 + T cell migration. Ccl2 increased Nf1 +/– CD8 + T cell migration relative to WT controls ( P < 0.0001). Nf1 +/- and WT CD8 + T cells treated with medium (Control) or Ccl12 exhibit similar migration. D, Nf1 +/- and WT CD4 + T cells had similar levels of Ccr4 expression. Nf1 +/– CD8 + T cells have increased Ccr4 expression relative to their WT counterparts ( P = 0.0474). E, Ccr4 inhibition (AZD2098; 15µM) in CD8 + T cells decreases Ccl2-induced, but not Ccl12-induced, migration ( P = 0.0063). F, Nf1 +/– and WT CD4 + T cells had similar levels of Ras activity. Nf1 +/– CD8 + T cells have increased Ras activity relative to their WT counterparts ( P = 0.0363) G, Inhibition of Ras activity (10µM IN-1) reduces Ccl2-induced,but not Ccl12-induced, CD8 + T cell migration ( P = 0.0104). H, RAS activity inhibition (10µM IN-1) decreases Ccr4 expression in Nf1+/- CD8 + T cells ( P = 0.0069). I, Proposed model of Ccl2 signaling through Ccr4 in Nf1+/– CD8 + T cells.

Article Snippet: 500 μl of chemoattractant media containing Ccl2 (20ng/ml, 479-JE-050/CF-R&D systems) or Ccl12 (25ng/ml, 428-P5-025-R&D systems) was added to the lower chamber , and the number of T cells in the lower chambers counted by microscopy at 20x after 6 h.

Techniques: Expressing, Migration, Control, Inhibition, Activity Assay