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: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: BMP9 inhibits CCL2 expression and release by endothelial cells. Confluent HPAECs were serum-restricted for 16 h followed by treatment with BMP9 in 0.1% FBS. (A) HPAECs were treated with 1 ng/ml BMP9 for 2, 4, 8 or 12 h (3 experiments). Data show the fold change relative to 0.1% FBS at each time point. (B) HPAECs were treated with BMP9 (0-10 ng/ml) for 8 h (5 experiments). (C) HPAECs were treated with BMP9 (0-10 ng/ml) for 24 h. CCL2 immunoreactivity of conditioned media was normalized to cell number for each well ( n =4 wells per treatment) and is representative of 3 experiments. (D) HPAECs were treated with BMP10 (0-10 ng/ml) for 8 h (3 experiments). (E) HPAECs were treated with BMP9 (1 ng/ml) or BMP10 (1 ng/ml) for 24 h. CCL2 immunoreactivity of conditioned media was normalized to cell number for each well. Data ( n =4 wells per treatment) are representative of 3 experiments. (F,G) HPAECs were treated with BMP9 (1 ng/ml) or BMP10 (1 ng/ml) for 8 h and expression of CCL2 (F) and ID1 and ID2 (G) measured (6 experiments). (H,I) HAECs were treated with BMP9 (0-10 ng/ml) (H) or BMP10 (0-10 ng/ml) (I) for 8 h (3 experiments). All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to control. Significance was calculated using either one-way repeated measures ANOVA with post-hoc Tukey's HSD test (B,E-G) or Friedman multiple comparison test with post-hoc Dunn's analysis (C,D,H,I). * P <0.05, ** P <0.01, *** P <0.001, compared with control (0.1% FBS without added BMP9 or BMP10).

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Expressing, Control, Comparison

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: Reduction of ALK1 attenuates the induction of CCL2 by BMP9, and both ACTR-II and BMPR-II mediate the repression by BMP9 and BMP10. (A-C) HPAECs were transfected with siRNA for ALK1 (siA1), BMPR2 (siB2) or a non-targeting control pool (siCP) using DharmaFECT1 (DH1). (A) HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 8 h. Expression of CCL2 was normalized to ACTB . Data show the fold change relative to DH1/0.1% FBS (4 experiments). (B) HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 24 h. Conditioned media were collected, assayed for CCL2 by ELISA and normalized to cell number for each well. Data ( n =4 wells per treatment) are from a representative of 4 experiments. (C) Specific reduction of ALK1 and BMPR-II by their respective siRNAs was confirmed by western blotting, the numbers below the blots representing band density ratios relative to α-tubulin normalised to the DH1 control. Arrows indicate the positions of the molecular mass markers (kDa). (D-F) HPAECs were transfected with a non-targeting control siRNA pool (siCP) or siRNAs for ACVR2A (siA2A), BMPR2 (siB2) or both in combination (siA2AB2) using DharmaFECT1 (DH1). HPAECs were treated with 1 ng/ml BMP9 or BMP10 in 0.1% FBS for 8 h. Expression of ACTR-IIA ( ACVR2A ; D), BMPR-II ( BMPR2 ; E) and CCL2 (F) were normalized to ACTB . Data show the fold change relative to DH1/0.1% FBS (6 experiments). The key for D-F is provided in F. (G-I) Confluent serum-restricted HPAECs were pretreated with 250 nM LDN-193189 or 2µM SD208 for 1 h followed by 1 ng/ml BMP9 in 0.1% FBS for 8 h for mRNA extraction. Expression of CCL2 (G), CXCL8 (H) and ID1 (I) were determined by qPCR and normalized to ACTB . qPCR data are presented as the fold change relative to the DMSO control (1:2500 in 0.1% FBS) (3 experiments). All data are expressed as mean±s.e.m. Significance was calculated using either a paired Students t -test (A,B,G-I), comparing with 0.1% FBS control, or one-way repeated measures ANOVA with post-hoc Sidak test (D-F), comparing with siCP of same treatment. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Transfection, Control, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Extraction

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: CCL2 repression by BMP9 in HPAECs is dependent on Smad4, but not Smad2 or Smad3. (A) Confluent serum-restricted HPAECs were treated with 0.01-10 ng/ml BMP9 in 0.1% FBS for 1 h. Protein lysates were immunoblotted for phospho-Smad1/5, Smad1, phospho-Smad2 or Smad2. Blots are representative of 4 experiments. (B) Quantification of the blots in A, calculated as the ratio of the density of the phospho-Smad band to the Smad band for each sample and normalised to the 0.1% FBS control. (C-E) HPAECs were transfected with SMAD4 siRNA (siS4) or a non-targeting control pool (siCP) using DharmaFECT1 (DH1). (C) Reduced Smad4 protein was confirmed by western blotting. (D) Confluent serum-restricted HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 8 h (3 experiments). (E) Confluent serum-restricted HPAECs were treated with 1 ng/ml BMP9 in 0.1% FBS for 24 h. CCL2 release was measured by ELISA and normalised to cell number. Data are from a representative of 3 experiments ( n =4 wells per treatment). (F,G) HPAECs were transfected with SMAD2 siRNA (siS2) or siCP using DH1 and treated with BMP9 for 8 h as described above. (F) CCL2 (top panel) and CXCL8 (bottom panel) expression (3 experiments). (G) Smad2 protein knockdown was confirmed by western blotting. (H,I) HPAECs were transfected with SMAD3 siRNA (siS3) or siCP using DH1 and treated with BMP9 for 8 h. (H) Smad3 protein knockdown was confirmed by western blotting. (I) CCL2 expression (3 experiments). For western blots, the migration positions of the relevant protein molecular mass markers (kDa) are indicated by arrows. The numbers below each blot panel represent band density ratios relative to α-tubulin normalised to the DH1 control. All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to DHI/0.1% FBS. Significance was calculated using one-way repeated measures ANOVA with post-hoc Tukey’s HSD test. * P <0.05.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Control, Transfection, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Knockdown, Migration

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: CCL2 repression by BMP9 in HPAECs is not dependent on Smad1, Smad5 or Smad9. HPAECs were transfected with siRNAs for SMAD1 (siS1), SMAD5 (siS5) or SMAD9 (siS9) alone or in combination using DharmaFECT1 (DH1). In parallel, cells were transfected with a non-targeting control pool (siCP). (A,B) Knockdown of Smad1 and Smad5 were confirmed by western blotting of cells transfected with siRNAs targeting individual (A) and combinations (B) of Smads. The migration positions of the relevant protein molecular mass markers (kDa) are indicated by arrows. The numbers below each blot panel represent band density ratios relative to α-tubulin normalised to the DH1 control. (C-E) Transfected HPAECs were serum-restricted, followed by treatment with 1 ng/ml BMP9 in 0.1% FBS for 8 h. CCL2 expression (C) and ID2 and CXCL8 expression (D) were determined in cells in which individual Smads were knocked down (5 experiments). CCL2 and ID2 expression (E) were determined in HPAECs in which combinations of Smads were knocked down (4 experiments). (F) Transfected HPAECs were serum-restricted, followed by treatment with 0.3 ng/ml BMP9 or BMP10 in 0.1% FBS for 4h (5 experiments). All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to DH1/0.1% FBS. Significance was calculated using one-way repeated measures ANOVA with post-hoc Tukey's HSD (C-E) or Sidak (F) test. * P <0.05, ** P <0.01, *** P <0.001. For CCL2 , data were compared with siCP/0.1% FBS. For ID2 and CXCL8 , data were compared with siCP/BMP9.

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Transfection, Control, Knockdown, Western Blot, Migration, Expressing

Journal: Journal of Cell Science

Article Title: Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells

doi: 10.1242/jcs.239715

Figure Lengend Snippet: BMP9 does not affect CCL2 induction by TNF-α in HPAECs and HAECs. (A) Confluent serum-restricted HPAECs were treated with BMP9 (5 ng/ml) alone or with TNF-α (5 ng/ml) in 0.1% FBS for 6 h. Co-treatments were added without BMP9 pre-incubation or after 1 h or 16 h pre-incubation with 5 ng/ml BMP9 (3 experiments). (B) Confluent serum-restricted HPAECs were treated with BMP9 (5 ng/ml) and TNF-α (5 ng/ml) in 0.1% FBS for 6 h. Conditioned media were assayed for CCL2 by ELISA. Data are from a representative of 3 experiments ( n =4 wells per treatment). (C) Confluent serum-restricted HPAECs were treated with BMP9 (0-5 ng/ml) and TNF-α (0-2 ng/ml) in 0.1% FBS for 6 h (4 experiments). (D,E) Confluent serum-restricted HAECs were treated with BMP9 (5 ng/ml) alone or with TNF-α (5 ng/ml) in 0.1% FBS for 6 h and assayed for CCL2 expression (D) (4 experiments) and CCL2 release (E). ELISA data are from a representative of 3 experiments ( n =4 wells per treatment). (F) Confluent serum-restricted HAECs were treated with BMP9 (0-5 ng/ml) and TNF-α (0-5 ng/ml) in 0.1% FBS for 6 h. All data are expressed as mean±s.e.m. Expression data are normalised to ACTB and presented as the fold change relative to 0.1% FBS (no additions). Significance was calculated using Friedman multiple comparisons test with post-hoc Dunn’s analysis. * P <0.05, ** P <0.01, *** P <0.001, compared with control (0.1% FBS).

Article Snippet: Samples (100 μl/well) and recombinant human CCL2 standards (3.9-2000 pg/ml; R&D Systems) were then added and incubated in a humidified chamber overnight at 37°C.

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Expressing, Control