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R&D Systems recombinant human ccl2 standards

BMP9 inhibits <t>CCL2</t> 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).

Recombinant Human Ccl2 Standards, supplied by R&D Systems, used in various techniques. Bioz Stars score: 99/100, based on 97 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Endothelial protective factors BMP9 and BMP10 inhibit CCL2 release by human vascular endothelial cells"

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

Journal: Journal of Cell Science

doi: 10.1242/jcs.239715

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).

Figure Legend 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).

Techniques Used: Expressing, Control, Comparison

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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.

Figure Legend 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.

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

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).

Figure Legend 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).

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

Image Search Results

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

Primer sequences used for RT-PCR and qPCR

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Evidence for a functional TSLP signalling axis in fibrotic lung disease

doi: 10.4049/jimmunol.1300588

Figure Lengend Snippet: Primer sequences used for RT-PCR and qPCR

Article Snippet: Paired antibodies MAB679 and BAF279 for the human CCL2 ELISA were obtained from R&D Systems and human recombinant CCL2 protein standard was from Peprotech (NJM, USA).

Techniques:

pHLFs were exposed to control medium (DMEM) or graded concentrations of TSLP for varying durations as designated. Time-course data (A) for the effect of TSLP (1 ng/ml) on pHLF CCL2 mRNA levels. CCL2 mRNA levels, at each designated time point, were assessed by qRT-PCR. Data are expressed as fold change for each time point relative to time zero, normalized to HPRT mRNA levels (mean ± SEM of triplicates from three independent experiments). (B and C) Time-course and concentration-response data for the effect of TSLP on pHLF CCL2 protein levels. pHLFs were exposed to TSLP (1 ng/ml) or control medium for varying durations (B) or graded concentrations of TSLP or control medium for 6 hours (C). CCL2 protein release into conditioned media was measured by ELISA and the amount of secreted CCL2 is expressed as pg/ml (mean ± SEM of triplicates from three independent experiments, *** p<0.001, comparison with time-point matched media control.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Evidence for a functional TSLP signalling axis in fibrotic lung disease

doi: 10.4049/jimmunol.1300588

Figure Lengend Snippet: pHLFs were exposed to control medium (DMEM) or graded concentrations of TSLP for varying durations as designated. Time-course data (A) for the effect of TSLP (1 ng/ml) on pHLF CCL2 mRNA levels. CCL2 mRNA levels, at each designated time point, were assessed by qRT-PCR. Data are expressed as fold change for each time point relative to time zero, normalized to HPRT mRNA levels (mean ± SEM of triplicates from three independent experiments). (B and C) Time-course and concentration-response data for the effect of TSLP on pHLF CCL2 protein levels. pHLFs were exposed to TSLP (1 ng/ml) or control medium for varying durations (B) or graded concentrations of TSLP or control medium for 6 hours (C). CCL2 protein release into conditioned media was measured by ELISA and the amount of secreted CCL2 is expressed as pg/ml (mean ± SEM of triplicates from three independent experiments, *** p<0.001, comparison with time-point matched media control.

Article Snippet: Paired antibodies MAB679 and BAF279 for the human CCL2 ELISA were obtained from R&D Systems and human recombinant CCL2 protein standard was from Peprotech (NJM, USA).

Techniques: Control, Quantitative RT-PCR, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison

TSLP induces STAT3 phosphorylation in pHLFs (A). Serum-starved pHLFs were exposed to TSLP (1 ng/ml) or control medium for the designated times and phosphorylation of STAT3 (p-STAT3) was assessed by Western blot analysis of total cell lysates using an anti-p-STAT3 antibody (A, upper panel). The same blot was stripped, re-probed with anti-total STAT3 antibody (A, lower panel), to verify protein loading. (B & C) The effect of S3I-201 on TSLP-induced STAT3 phosphorylation and CCL2 protein release. pHLFs were pre-incubated with graded concentrations of S3I-201 for 30 minutes prior to exposure to TSLP (1 ng/ml) or control medium for the designated times. Final concentrations of DMSO were kept constant for all experimental conditions (0.1% in DMEM). (B) p- and t-STAT3 following exposure to TSLP for 30 mins was assessed as above. (C) CCL2 protein release into conditioned media at 6 hours was measured by ELISA. Data are presented as a percentage of the maximal response obtained with TSLP and drug vehicle alone. The first bar represents the CCL2 response to TSLP and drug vehicle alone. The second bar represents the response to control medium alone. The third bar represents highest concentration of S3I-201 examined and shows that this compound has no effect on basal CCL2 release. Negative log of the concentrations of S3I-201 are presented. Data represent the mean ± SEM of triplicates from three independent experiments. +++ p<0.001, comparison with untreated cells; * p<0.05; *** p<0.001, comparison with TSLP alone. (D, E & F) The effect of siRNA knockdown of STAT3 expression on TSLP-induced CCL2 protein release by pHLFs. pHLFs were transfected with siRNA targeting STAT3, scrambled siRNA (final siRNA concentration 100nM) or mock-transfected. Cells were then exposed to TSLP (1 ng/ml) or control medium for 6 hours before total cell lysates were prepared for Western blot analysis (D & E); matched conditioned media was also removed for CCL2 protein measurements by ELISA (F). Knockdown of STAT3 expression was assessed by Western blot analysis of total cell lysates from cells exposed to TSLP (1 ng/ml) (D) using an anti-t-STAT3 antibody (upper panel). The same blots were stripped, re-probed with an anti-total ERK1/2 antibody (lower panel) and used to verify protein loading. Densitometric analysis was performed on total STAT3 expression (E) and data are presented as the mean ratio of total STAT3 over total ERK2, normalised to mock-transfected control, Data represent the mean ± SEM of triplicates from three independent experiments Data represent the mean ± SEM of triplicates from three independent experiments. (F) CCL2 protein release into conditioned media was measured by ELISA and data are presented as pg/ml (mean ± SEM of triplicates from three independent experiments). *** p < 0.001, comparison with mock-transfected cells.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Evidence for a functional TSLP signalling axis in fibrotic lung disease

doi: 10.4049/jimmunol.1300588

Figure Lengend Snippet: TSLP induces STAT3 phosphorylation in pHLFs (A). Serum-starved pHLFs were exposed to TSLP (1 ng/ml) or control medium for the designated times and phosphorylation of STAT3 (p-STAT3) was assessed by Western blot analysis of total cell lysates using an anti-p-STAT3 antibody (A, upper panel). The same blot was stripped, re-probed with anti-total STAT3 antibody (A, lower panel), to verify protein loading. (B & C) The effect of S3I-201 on TSLP-induced STAT3 phosphorylation and CCL2 protein release. pHLFs were pre-incubated with graded concentrations of S3I-201 for 30 minutes prior to exposure to TSLP (1 ng/ml) or control medium for the designated times. Final concentrations of DMSO were kept constant for all experimental conditions (0.1% in DMEM). (B) p- and t-STAT3 following exposure to TSLP for 30 mins was assessed as above. (C) CCL2 protein release into conditioned media at 6 hours was measured by ELISA. Data are presented as a percentage of the maximal response obtained with TSLP and drug vehicle alone. The first bar represents the CCL2 response to TSLP and drug vehicle alone. The second bar represents the response to control medium alone. The third bar represents highest concentration of S3I-201 examined and shows that this compound has no effect on basal CCL2 release. Negative log of the concentrations of S3I-201 are presented. Data represent the mean ± SEM of triplicates from three independent experiments. +++ p<0.001, comparison with untreated cells; * p<0.05; *** p<0.001, comparison with TSLP alone. (D, E & F) The effect of siRNA knockdown of STAT3 expression on TSLP-induced CCL2 protein release by pHLFs. pHLFs were transfected with siRNA targeting STAT3, scrambled siRNA (final siRNA concentration 100nM) or mock-transfected. Cells were then exposed to TSLP (1 ng/ml) or control medium for 6 hours before total cell lysates were prepared for Western blot analysis (D & E); matched conditioned media was also removed for CCL2 protein measurements by ELISA (F). Knockdown of STAT3 expression was assessed by Western blot analysis of total cell lysates from cells exposed to TSLP (1 ng/ml) (D) using an anti-t-STAT3 antibody (upper panel). The same blots were stripped, re-probed with an anti-total ERK1/2 antibody (lower panel) and used to verify protein loading. Densitometric analysis was performed on total STAT3 expression (E) and data are presented as the mean ratio of total STAT3 over total ERK2, normalised to mock-transfected control, Data represent the mean ± SEM of triplicates from three independent experiments Data represent the mean ± SEM of triplicates from three independent experiments. (F) CCL2 protein release into conditioned media was measured by ELISA and data are presented as pg/ml (mean ± SEM of triplicates from three independent experiments). *** p < 0.001, comparison with mock-transfected cells.

Article Snippet: Paired antibodies MAB679 and BAF279 for the human CCL2 ELISA were obtained from R&D Systems and human recombinant CCL2 protein standard was from Peprotech (NJM, USA).

Techniques: Phospho-proteomics, Control, Western Blot, Incubation, Enzyme-linked Immunosorbent Assay, Concentration Assay, Comparison, Knockdown, Expressing, Transfection

Conditioned media (CM) from pHLFs exposed to TSLP induces chemotaxis of human THP-1 monocytes (A). Human THP-1 monocytes were exposed to CM collected from pHLFs treated with graded concentrations of TSLP (0-1 ng/ml) for 6 hours; chemotaxis was assessed using a Boyden chamber as described in Materials and Methods. The first bar represents the chemotactic response of THP-1 cells to DMEM/BSA (1%, w/v) only. The second bar represents the chemotactic response to rhCCL2. Data show the mean number of cells per 5 high powered fields (HPF) ± SEM of triplicates from three independent experiments. * p<0.05, comparison with conditioned media from unstimulated cells. TSLP-induced CCL2 mediates monocyte chemotaxis (B). THP-1 cells were exposed to CM from pHLFs (treated with 1 ng/ml TSLP) which had been pre-incubated with an anti-CCL2 neutralizing antibody (Ab) or isotype control (IC) antibody (both at 30 μg/ml) and chemotaxis assessed as above. The first bar represents the chemotactic response of cells to recombinant human (rh) TSLP (1 ng/ml). Data are presented as above. * p<0.05, comparison with CM from pHLFs exposed to TSLP only.

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Evidence for a functional TSLP signalling axis in fibrotic lung disease

doi: 10.4049/jimmunol.1300588

Figure Lengend Snippet: Conditioned media (CM) from pHLFs exposed to TSLP induces chemotaxis of human THP-1 monocytes (A). Human THP-1 monocytes were exposed to CM collected from pHLFs treated with graded concentrations of TSLP (0-1 ng/ml) for 6 hours; chemotaxis was assessed using a Boyden chamber as described in Materials and Methods. The first bar represents the chemotactic response of THP-1 cells to DMEM/BSA (1%, w/v) only. The second bar represents the chemotactic response to rhCCL2. Data show the mean number of cells per 5 high powered fields (HPF) ± SEM of triplicates from three independent experiments. * p<0.05, comparison with conditioned media from unstimulated cells. TSLP-induced CCL2 mediates monocyte chemotaxis (B). THP-1 cells were exposed to CM from pHLFs (treated with 1 ng/ml TSLP) which had been pre-incubated with an anti-CCL2 neutralizing antibody (Ab) or isotype control (IC) antibody (both at 30 μg/ml) and chemotaxis assessed as above. The first bar represents the chemotactic response of cells to recombinant human (rh) TSLP (1 ng/ml). Data are presented as above. * p<0.05, comparison with CM from pHLFs exposed to TSLP only.

Article Snippet: Paired antibodies MAB679 and BAF279 for the human CCL2 ELISA were obtained from R&D Systems and human recombinant CCL2 protein standard was from Peprotech (NJM, USA).

Techniques: Chemotaxis Assay, Comparison, Incubation, Control, Recombinant

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