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

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: Scientific reports

Article Title: Anti-CCL2 antibody combined with etoposide prolongs survival in a minimal residual disease mouse model of neuroblastoma.

doi: 10.1038/s41598-023-46968-2

Figure Lengend Snippet: Figure 3. CCL2 expression from neuroblastoma cells and monocytes. (A) Utilizing four neuroblastoma cell lines (SH-SY5Y, CHLA-255, NGP, SMS-KCNR), neuroblastoma cells were co-cultured with monocytes, and CCL2 protein expression was measured by ELISA. Co-culture of neuroblastoma cells and monocytes leads to higher CCL2 expression compared to either alone. (B) To determine the primary source of CCL2 protein expression, monocytes were co-cultured with neuroblastoma supernatant and neuroblastoma cells were co-cultured with monocyte supernatant. Monocytes appear to contribute more CCL2 expression than neuroblastoma cells. Experiments were performed in quadruplicate for each cell line. Student’s t-test of log 10 transformed data was performed; errors bars represent mean ± SEM. The effect of CCL2 and anti-CCL2 antibody on neuroblastoma cells. (C) Cytotoxicity assays were performed on three neuroblastoma cell lines (SH-SY5Y-Fluc, CHLA-255-Fluc, NGP-Fluc) and incubated for 24 h and 48 h (D) in varying concentrations of recombinant CCL2, with and without anti-CCL2 antibody. The addition of CCL2, with and without anti-CCL2 antibody, did not affect neuroblastoma cell survival (p > 0.5 for all). ANOVA was performed; bar graphs and errors bars represent mean ± SEM.

Article Snippet: Recombinant human CCL2 protein was obtained from R&D Systems (Minneapolis, MN, USA, Cat # 279-MC050).

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Co-Culture Assay, Transformation Assay, Incubation, Recombinant

Journal: Scientific reports

Article Title: Anti-CCL2 antibody combined with etoposide prolongs survival in a minimal residual disease mouse model of neuroblastoma.

doi: 10.1038/s41598-023-46968-2

Figure Lengend Snippet: Figure 5. Combination therapy of anti-CCL2 antibody and etoposide improves survival in mice following surgical resection of primary tumors created from CHLA-255-Fluc (A), NGP-Fluc (B), and PDX (COG-N- 415X) (C). Kaplan–Meier survival plots shown here; differences in survival between groups were analyzed with linear regression.

Article Snippet: Recombinant human CCL2 protein was obtained from R&D Systems (Minneapolis, MN, USA, Cat # 279-MC050).

Techniques:

Journal: Scientific reports

Article Title: Anti-CCL2 antibody combined with etoposide prolongs survival in a minimal residual disease mouse model of neuroblastoma.

doi: 10.1038/s41598-023-46968-2

Figure Lengend Snippet: Figure 6. Combination treatment of etoposide with anti-CCL2 Ab decreases tumor burden in NOD-SCID gamma mice (NSG) injected with neuroblastoma (CHLA-255-Fluc and NGP-Fluc) in a minimal residual disease mouse model of neuroblastoma. (A) Following tumor resection of CHLA-255-Fluc orthotopic xenografts, mice were divided into four treatment groups (control, anti-CCL2 antibody alone, etoposide alone, and combination of etoposide and anti-CCL2 antibody) and underwent weekly bioluminescent imaging to measure tumor burden over time. (B) Combination therapy of anti-CCL2 Ab and etoposide led to significantly decreased tumor burden compared to untreated control. (C) Repeat experiment utilizing the NGP-Fluc neuroblastoma cell line. (D) Combination therapy of anti-CCL2 Ab and etoposide demonstrated decreased tumor burden compared to control. Interval regression was performed and line graphs with errors bars represent mean ± SD.

Article Snippet: Recombinant human CCL2 protein was obtained from R&D Systems (Minneapolis, MN, USA, Cat # 279-MC050).

Techniques: Injection, Control, Imaging

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: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 increases cell surface expression of P2X4R protein in microglial cells. P2X4R protein on the surface of primary cultured microglial cells was detected by cell surface biotinylation followed by Western blot analysis of P2X4R protein using its specific antibody. Total cellular P2X4R protein was detected in whole-cell lysates of microglial cells. Microglial cells in primary cultures were treated with either CCL2 (100 ng/mL), CX3CL1 (100 ng/mL), or IFN-γ (100 U/mL) for 30 min (a) with CCL2 (10, 30, and 100 ng/mL) for 30 min (b) and with CCL2 (100 ng/mL) for 30, 60, and 120 min (c). b, c histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four to five times, and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05 compared with unstimulated control microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Cell Culture, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCR2 mediates CCL2-induced surface P2X4R expression. a–c Microglial cells in primary cultures were pretreated with either CCL2 neutralizing antibodies (CCL2-Ab, 2.5 and 25 μg/mL) for 30 min (a), RS-504393 (RS, 1 and 10 μM) for 20 min (b) or forskolin (10 μM) for 10 min (c) before CCL2 (100 ng/mL) stimulation. d Microglial cells were stimulated by CCL12 (10 ng/mL) for 30 min. Surface proteins of microglial cells were biotinylated 30 min after CCL2 (a–c) and CCL12 (d) stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four to five times, and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, **P < 0.01, ***P < 0.001 compared with unstimulated control microglia; #P < 0.05, ##P < 0.01 compared with CCL2-stimulated microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 is involved in fibronectin-induced surface P2X4R upregulation. a Biotinylation assay of surface P2X4R proteins. CCL2 neutralizing antibody (CCL2-Ab, 25 μg/mL) and normal goat IgG (IgG, 25 μg/mL) were pretreated for 30 min before fibronectin (FN, 10 μg/mL) stimulation. Surface proteins of microglial cells were biotinylated 24 h after fibronectin stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated five times and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, **P <0.01 compared with unstimulated control microglia; #P < 0.05 compared with fibronectin-stimulated microglia. b CCL2 levels in supernatants of microglial cultures were measured by ELISA; primary cultured microglial cells were treated with or without fibronectin (10 μg/mL) for various time points of testing. The experiments were repeated six times, and the data are means ± SEM of the concentration of CCL2 (pg/mL). ***P < 0.001 compared with control at 1 h

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Cell Surface Biotinylation Assay, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Concentration Assay

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 enhances movement of P2X4Rs. a Immunocytochemical analysis of P2X4Rs detected by its specific antibody. Primary cultured microglia were stimulated by CCL2 (100 ng/mL) for 30 min and were fixed by formaldehyde. Cells were pretreated with CCL2 neutralizing antibody (CCL2-Ab, 25 μg/mL) and forskolin (10 μM) for 30 and 10 min, respectively, before CCL2 stimulation. b Monitoring of P2X4R-GFP fluorescence particles in living microglial cells transduced with a lentiviral vector encoding P2X4R-GFP. Left Typical trajectories of P2X4R-GFP fluorescence particles in microglia treated with HBSS (control) or CCL2 (100 ng/mL). Right The total distance (μm, means ± SEM, n = 19) of the trajectories of P2X4R-GFP fluorescence particles from initial position. ***P < 0.001 compared with the control group by two-way ANOVA. Scale bar: 10 μm

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Cell Culture, Fluorescence, Transduction, Plasmid Preparation, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: Dynamin inhibitor does not affect the effect of CCL2 on cell surface P2X4R expression. Microglial cells in primary cultures were pretreated with the dynamin inhibitor dynasore (80 μM) for 30 min before CCL2 (100 ng/mL) stimulation. Surface proteins of microglial cells were biotinylated 30 min after CCL2 stimulation. Surface and total P2X4R proteins were detected by Western blot analysis using its specific antibody. Histograms of the relative band density ratio of surface and total P2X4R protein normalized to the levels of β-actin. The experiments were repeated four times and the data are means ± SEM of fold changes over the value of unstimulated control microglia. *P < 0.05, ***P < 0.001 compared with unstimulated control microglia

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Expressing, Western Blot, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 causes lysosomal exocytosis in microglial cells. a Immunofluorescence for P2X4R (green) and LGP85 (red), a marker of lysosome, in primary cultured microglial cells treated with or without CCL2 (100 ng/mL) for 30 min. b The release of the lysosomal enzyme β-hexosaminidase from microglial cells was assayed by measuring its activity in the supernatants of microglial cultures collected at 30 min after CCL2 (100 ng/mL) or ionomycin (5 μM) stimulation. The experiments were repeated six times and the data are means ± SEM. *P < 0.05, **P < 0.01 compared with unstimulated control microglia. Scale bar: 10 μm

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Immunofluorescence, Marker, Cell Culture, Activity Assay, Control

Journal: Purinergic Signalling

Article Title: CCL2 promotes P2X4 receptor trafficking to the cell surface of microglia

doi: 10.1007/s11302-011-9288-x

Figure Lengend Snippet: CCL2 enhances P2X4R-mediated Akt phosphorylation in microglial cells. Primary cultured microglia were treated with or without CCL2 (100 ng/mL) for 30 min. Western blot analysis of the levels of phosphorylated and total Akt protein in whole-cell lysate of CCL2-treated microglia 10 min after ATP (5 μM) stimulation. Cells were pretreated with TNP-ATP (100 μM) 5 min before ATP stimulation. A histogram of the relative band density ratio of Akt phosphorylation normalized to the levels of β-actin. The experiments were repeated five times, and the data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: Microglial cells were stimulated by applying either recombinant rat CCL2 (10, 30, or 100 ng/mL; R&D Systems, Lille, France), mouse CCL12 (10 ng/mL; R&D Systems), rat CX3CL1 (100 ng/mL; R&D Systems), or rat IFN-γ (100 U/mL; Calbiochem, San Diego, CA, USA) 30 min before biotinylation.

Techniques: Phospho-proteomics, Cell Culture, Western Blot

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: (A) Uniform manifold approximation and projection (UMAP) plot showing identified endothelial cell types in lungs from 3 IPAH patients and 6 donor controls (GSE169471). (B) Venn diagram showing the overlap of upregulated genes in SU-Hx rat lung (SU-Hx–Isotype control vs. Nx), downregulated genes in Ab93-treated SU-Hx rat lung (SU-Hx–Ab93 vs. SU-Hx–Isotype), and upregulated genes in IPAH pulmonary general capillary endothelial cells (gCap) (IPAH vs. Control, GSE169471). (C) Venn diagram showing the overlap of upregulated genes in BMP9-treated HPMVEC (BMP9-1.5h vs. control) and upregulated genes in IPAH pulmonary gCap ECs (IPAH vs. Control, GSE169471). (D-G) Volcano plots showing changes and significance of candidate driver genes across different datasets, including (D) upregulation of ID1 , BMPR2 , CXCL12, EDN1, COL18A1 , and IGFBP4 in IPAH vs. control lungs; (E) upregulation of ID1, BMPR2 , EDN1, IGFBP4, VEGFA, PDGFA, PDGFB, VWA1 , and SOX18 in BMP9-treated PMVEC; (F) upregulation of Edn1, Cald1, Vwa1, Col18a1 , and Igfbp4 in Su-Hx treated rats vs. normoxia; and (G) downregulation of Cxcl12, Igfbp4, Col18a1, Vwa1 , and Cald1 in Ab93-treated vs. isotype control treated SU-Hx rats. (H-L) Violin plots showing increased expression of CXCL12 , IGFBP4 , EDN1 , ENG , and BMPR2 in various subsets of endothelial cells from IPAH patients (GSE169471). (M) BMP9 (40 pM) increased CXLC12 mRNA expression in HPMVEC in a time-dependent manner. (N-R) BMP9 (40 pM) increased secretion of CXCL12, IGFBP4, ET-1, PDGF-BB and CCL2 in cultured HPMVEC supernatants. ( n = 3 per group, means ± SD. *P<0.05 as compared to 0 h or all the other groups, Dunnett’s test) (S-U) BMP9 (40 pM) treatment of HPMVEC increased mRNA expression of CXCL12 , which was diminished when BMPR2 , ACVRL1 or ENG were knocked down. ( n = 3 per group, mean ± SD, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001 by one-way ANOVA with Sidak’s test).

Article Snippet: To test the concentration of CXCL12, ET1, PDGF-BB, and CCL2 in PMVECs culture medium, the Ella Simple Plex system and the cartridges (Human CXCL12, SPCKB-PS-000299; Human Endothelial-1, SPCKC-PS-000265; HumanCCL2 and PDGF-BB, SPCKC-PS-006510; Protein Simple) were used according to the manufacturer’s instructions.

Techniques: Control, Expressing, Cell Culture

Journal: bioRxiv

Article Title: BMP9 regulates the endothelial secretome to drive pulmonary hypertension

doi: 10.1101/2025.08.29.673113

Figure Lengend Snippet: ( A ) Dot plot highlighting log 10 average expression of selected marker genes used to identify endothelial clusters. The dot size corresponds to the percentage of cells expressing a gene in a given cluster. ( B ) Venn diagram showing the overlap of upregulated genes in SU-Hx rat lung (SU-Hx– Isotype control vs Nx), downregulated genes in Ab93-treated SU-Hx rat lungs (SU-Hx–Ab93 vs SU-Hx–Isotype), and upregulated genes in IPAH pulmonary artery endothelial cells (AEC, IPAH vs Control, GSE169471). ( C ) Venn diagram showing the overlap of upregulated genes in BMP9-treated HPMVEC (BMP9-1.5h vs control) and upregulated genes in IPAH pulmonary AECs (IPAH vs Control, GSE169471). ( D ) Volcano plot showing potential candidate genes upregulated in IPAH pulmonary AEC (IPAH vs Control, GSE169471). BMP9 (40 pM) increased CXLC12 mRNA ( E ) and ( F ) protein expression in HPAEC at varying intervals up to 24h. Stimulation of cultured HPAEC with BMP9 (40 pM) increased ( G ) IGFBP4 mRNA and ( H ) protein expression, as well as protein expression of ( I ) Endothelin-1, ( J ) PDGF-BB, and ( K ) CCL2 protein in supernatants at varying intervals over 24h. ( n = 3 per group, mean ± SD, *P<0.05 as compared to 0 h, one-way ANOVA with Dunnett’s multiple comparisons test).

Article Snippet: To test the concentration of CXCL12, ET1, PDGF-BB, and CCL2 in PMVECs culture medium, the Ella Simple Plex system and the cartridges (Human CXCL12, SPCKB-PS-000299; Human Endothelial-1, SPCKC-PS-000265; HumanCCL2 and PDGF-BB, SPCKC-PS-006510; Protein Simple) were used according to the manufacturer’s instructions.

Techniques: Expressing, Marker, Control, Cell Culture

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Integrated analysis of chemokine/cytokine protein after 9-Gy myeloablative radiation, HSCs, and adoptive cellular therapy and in vitro migration assays and single-cell RNA sequencing analysis in KR158B glioma. (A) Cytokine and chemokine profiling of glioma protein lysates in the tumor secretome in adoptive cellular therapy–treated gliomas and untreated control. Statistical comparisons were made using the 1-way ANOVA test. (B) CCL12 protein levels in glioma lysates were quantified by ELISA. Statistical analysis was made using the unpaired Student t test. (C) Transcriptomic analysis of chemokine ligands and receptors using nanoString digital spatial profiling. Statistical analysis was performed using the 2-way ANOVA test. (D) In vitro trans-well migration assay of MDSCs migrating in response to glioma conditioned media from excised glioma tissue combined with neutralizing antibodies against CCL2 and/or CCL12. Statistical analysis was performed using the 1-way ANOVA test. (E) ELISA CCL12 protein quantification using immune cell–specific isolates from excised glioma tissue. Statistical analysis was performed using the 1-way ANOVA test. (F) Immune cell subset deconvolution from single-cell RNA sequencing analysis from excised glioma tissue. (G) mRNA expression of CCL12 overlayed on immune cell subset deconvolution using single-cell RNA sequencing analysis from excised glioma tissue. (H) In vitro trans-well migration assay of MDSCs migration in response to immune cell-specific isolates combined with either IgG or CCL12 neutralizing antibody. Statistical comparisons conducted using the 2-way ANOVA test. * P ≤ .05. ** P ≤ .01. *** P ≤ .001. **** P ≤ .0001; error bars: mean ± standard deviation. Abbreviations: ANOVA, analysis of variance; MDSCs, myeloid-derived suppressor cells.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: In Vitro, Migration, Single Cell, RNA Sequencing, Control, Enzyme-linked Immunosorbent Assay, Expressing, Standard Deviation, Derivative Assay

Journal: Neuro-Oncology Advances

Article Title: Adoptive cellular therapy prevents reconstitution of myeloid-derived suppressor cells in the glioma tumor microenvironment

doi: 10.1093/noajnl/vdag054

Figure Lengend Snippet: Association of Ccl2 and macrophage-associated gene expression with clinical outcomes in glioblastoma patients. (A) Kaplan-Meier analysis of disease-free survival based on Ccl2 expression. (B) Kaplan-Meier analysis of overall survival of Cd14 expression. (C) Kaplan-Meier analysis of disease-free survival stratified by Fcgr3a expression. (D) Deconvolution of single-cell RNA sequencing cell subsets in the glioma microenvironment of GBM patients. (E) Single-cell RNA sequencing deconvolution of Ccl2 expression among cell clusters from GBM patients. (F) Deconvoluted quantification of Ccl2 expression from cell clusters identified from GBM patient samples. Statistical comparisons made using the log-rank test with P values presented on the survival graphs along with hazard ratios and P values for hazard ratios.

Article Snippet: Recombinant mouse CCL2 (R&D Systems, cat. 479-JE) and CCL12 (R&D Systems, 428-P5) were diluted in the migration buffer and plated at 150 μL/well in the lower Boyden chamber.

Techniques: Gene Expression, Expressing, Single Cell, RNA Sequencing