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