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: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Inflammatory monocytes associate with survival in LUSC. a Kaplan–Meier plots of overall survival in lung squamous carcinoma (LUSC) patients split by median (left, p < 0.0001) and quartile (right, p = 0.0006) expression levels of CD14. P -values are obtained with the log-rank test; FDR were calculated according to Benjamini and Hochberg. b Proportion of patients by mRNA subtype that have CD14 expression levels above (red) or below (black) the median CD14 expression level. Binomial tests for proportions were performed. (Black asterisks: significant enrichment below the median, red asterisks: Significant enrichment above the median). c Kaplan–Meier plots of overall survival in LUSC by expression levels of CCL2 ( p = 0.001), CCL3 ( p = 0.018) and CSF1 ( p = 0.015) expression. d Pearson’s correlations of CCL2, CCL3, and CSF1 chemokines versus CD14 (gene expression). e Dynamic range of mRNA expression of CD14 and CCL2 for each LUSC mRNA subtype. P -values were obtained with analysis of variance. The purple shading for CD14 expression represents samples in the ‘IM-rich subset’ (above the median CD14 expression level). f Dynamic range of mRNA expression of CCL3 and CSF1 by LUSC mRNA subtype. g Representative multiplex IHC for CD14 (green), CCR2 (red) and pan-cytokeratin (light blue) in a LUSC tumor sample, and enumeration of CD14+/CCR2+ cells in CK+ and CK- regions. #1-3 represent CK- regions, #4 represents a CK + region. Scale bar 100 μm. h Representative dual CD14+/CCR2+ cells (white arrows) in CK- (#1-3) and CK+ (#4) regions. Note: CK + region shown in white channel to more easily appreciate green and red. Scale bar 25 μm. i Enumeration of CD14+/CCR2+ cells in CK- and CK+ regions by mRNA subtype. Classical ( n = 14), Basal ( n = 9), Primitive ( n = 6) and Secretory ( n = 12). p -values were obtained with Student’s t-test in comparison to the Classical subtype. * P < 0.05, ** P < 0.01, *** P < 0.0001

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Expressing, Gene Expression, Multiplex Assay, Comparison

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: TNFα activation of NFκB promotes CCL2-mediated IM recruitment. a Microarray expression data (left) comparing murine bronchial epithelial cells (MBECs), parental KLN205 and the LN4K1 sub-clone. Top upstream pathways (right) from Ingenuity Pathway Analysis (IPA) are shown for the differentially regulated genes shown in brackets. b An upstream network visualization from IPA of all over-expressed genes (all nodes) in the upper portion of the heat map shown in Fig. with significant log-rank (survival analysis) p -value ( < 0.05). TNFα and NFκB (blue nodes) were amongst the top upstream regulators known to have direct roles (black lines) in promoting CCL2, CCL3 and CSF1 chemokines (the degree of redness corresponds with increasing statistical significance). c Relative expression of TNFα. d CCL2, CCL3 and CSF1 by qPCR. Data are averages ± s.e.m. P -values were obtained with Student’s t-test in comparison with KLN205. e Relative levels of CCL2 as measured by ELISA from secreted media of cells growing in vitro or, f , from plasma of tumor-bearing mice. Data are averages ± s.e.m. g Relative mRNA expression of CCL2 and p65 in LN4K1 cells following treatment with control or p65 siRNA with or without exogenous TNFα (100 ng/mL). h Relative expression of CCL2 mRNA (top) and phospho-p65 and p65 protein (bottom) following treatment with DMSO or an IKKβ inhibitor (Compound A, 5 μM) for 5 h. i Relative IM counts in the bone marrow, blood, spleen from healthy DBA2 mice versus those with LN4K1 tumors. j Relative IM, TAM and TReg counts from the lungs of healthy versus LN4K1-bearing mice. IMs were also assessed in age-matched DBA2 mice following HBSS ‘Mock’ injection. P -values obtained with one-sided Student’s t-test, n = 5 mice/group for f , i , and j . * P < 0.05, ** P ≤0.01, *** P ≤0.001

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Activation Assay, Microarray, Expressing, Comparison, Enzyme-linked Immunosorbent Assay, In Vitro, Clinical Proteomics, Control, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: CCL2-mediated IM recruitment is critical for LUSC metastasis. a Relative expression of CCL2 for KLN205 and sub-clones. b Quantification of luciferase signal. c Representative images obtained 10 days after cell injection of (i) KLN205-Scr ORF, (ii) KLN205-CCL2 ORF, (iii) LN4K1-Cntrl shR, (iv) LN4K1-CCL2 shR#1 and (v) LN4K1-CCL2 shR#2. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. d Survival plots of mice following tail vein injection of KLN205 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. e Number of IMs per lung lobe, n = 12 lobes/group. f Survival plots of mice following tail vein injection of LN4K1 cell lines. The black arrow indicates tissue harvest, n = 10 mice/group. g Number of IMs per lung lobe, n = 12 lobes/group. h Schematic (left) and quantification of luciferase signal (right) of mice treated with vehicle or PF-04136309 to assess effects on established metastases. i FACS plots and ( j ) quantification of percent IMs in the blood and ( k ) right lung of LN4K1-bearing mice. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 5 mice/group. l , FACS analysis of percent immune infiltrates for TAMs (gated on F480), DCs (gated on SiglecF-/CD11c), CD4, CD8, Tregs (gated on TCRb + ) and NK cells (gated on SiglecF-/B220-/TCRb-). m Schematic (left) and quantification of luciferase signal (right) of mice treated at the time of cell injection to assess effects on preventing metastasis. Data are averages ± s.e.m. P -values were obtained with Student’s t-test, n = 10 mice/group. n.s. = non-significant, * P ≤0.05, *** P < 0.001. For panel b , * FDR < 0.05, ** FDR < 0.01

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Expressing, Clone Assay, Luciferase, Injection

Journal: Nature Communications

Article Title: Factor XIIIA—expressing inflammatory monocytes promote lung squamous cancer through fibrin cross-linking

doi: 10.1038/s41467-018-04355-w

Figure Lengend Snippet: Schematic of the ‘IM-rich subset’ of lung squamous carcinoma. TNFα activation of the canonical NFκB leads to LUSC cell secretion of chemo-attractant CCL2, which stimulates the bone marrow to release inflammatory monocytes (IMs) into circulation. The IMs bring large payloads of FXIIIA into the tumor microenvironment, leading to cross-linked fibrin, LUSC invadopodia formation and progression

Article Snippet: Murine CCL2 protein levels were quantified by ELISA using the DuoSet Immunoassay kit (R&D Systems DY479-05 and DY008) according to the manufacturer’s protocol.

Techniques: Activation Assay