Fig 2: The protein expressions of Smad1 and p-Smad1/5/8 in the heart and aorta (n = 8 for each group, A). The protein expressions of Smad1 (B and C) and p-Smad1/5/8 (D and E) were significantly reduced in the heart (B and D) and aorta (C and E) in the ob/ob+ BMP4 shRNA group compared with that in the ob/ob+ Lac Z shRNA group (*P < 0.05). No statistically significantly different was observed between ob/ob+ BMP4 shRNA, C57BL/6+Lac Z shRNA, and C57BL/6+BMP4 shRNA groups (P > 0.05).

Fig 3: Activation of the Rho signaling pathway inhibits BMP-2-induced Smad1 expression in PAH. PAH-PASMCs were cultured and treated with 10 ng/ml Rho activator PDGF-BB, 2 ng/ml Smad1 activitor BMP-2, 10 µM ROCK inhibitor Y-27632 and 20 µM MEK1 inhibitor U0126. Immunofluorescence staining results were measured at 0–210 min of the experiment. Or 12 h following treatment with final BMP-2, total and nuclear proteins were extracted, respectively. (A) Representative confocal immunofluorescent staining micrographs of distribution of p-Smad1 (Ser463/465) 60 min after exposure to activator and inhibitor in PASMCs. The results were analyzed quantitatively. *P<0.05; Scale bar, 200 µm. (B) Changes of the percentage of cells positive for p-Smad (Ser463/465) nuclear accumulation after activator and inhibitor exposure in PAH-PASMCs. *P<0.05. the BMP-2 group vs. the untreated group; #P<0.05 the BMP-2+PDGF-BB group vs. the BMP-2 group; &P<0.05 the BMP-2+PDGF-BB+Y27632 or the BMP-2+PDGF-BB+U0126 group vs. the BMP-2+PDGF-BB group. (C) Total Smad1, total p-Smad1 (Ser206) and nuclear p-Smad1 (Ser463/465) protein levels following activation of the Rho and BMP-2 signaling pathways in PASMCs from rat models of PAH were measured by western blotting. *P<0.05. Data are presented as the mean ± standard deviation, n=3. PAH, pulmonary artery hypertension; PASMC, pulmonary artery smooth muscle cell; BMP-2, bone morphogenetic protein 2; PDGF-BB, platelet derived growth factor-BB; Smad1, mothers against decapentaplegic homolog 1; p, phosphorylated.

Fig 4: Expression of TGF-β/BMP pathway component proteins are dysregulated in AVM tissue. Protein levels of TGF-β1, ALK1, ALK5, and BMP-9 (a) and of Smad1–8 (c) were analyzed by western blot from AVM tissue samples (n = 10 patients) and normal tissue samples (n = 10 patients). Representative examples from each group are illustrated. GAPDH and α-tubulin protein expression were determined as loading controls. The total density of each protein band (n = 10 AVM and n = 10 normal) was determined using an ImageQuant developer and software, and relative expression was calculated by normalizing to loading controls (b,d). Error bars represent standard deviation. Paired t tests were performed to determine statistical significance (asterisks: *P < 0.05, **P < 0.01, ***P < 0.001).

Fig 5: BMP9 activates the Smad1,5,8 pathway in HepG2 cells. A. HepG2 cells were incubated for 1 hour with different concentrations of BMP9 in 0.1% FBS media. Western blots were performed with antibodies that recognized activated (phosphorylated) Smad1, 5 and 8 (P-Smad1,5,8) and Smad1 as loading control. A representative experiment of 2 is shown. B. HepG2 cells were incubated for different periods of time −/+ BMP9 (5 ng/ml) in 0.1% FBS media. Western blots were performed with antibodies that recognize Id1, P-Smad1,5,8 and total Smad1 (loading control). A representative experiment of 3 is shown. C. HepG2 stably expressing BRE-luciferase (HepG2-BRA) cells were plated and incubated with 0.1% FBS for 15 hours, and then treated with different concentrations of BMP9 for additional 15 hours. Luciferase activity was normalized to cell number. Data are shown as fold induction (relative to untreated cells) and are from one representative experiment (n = 4) out of 3 performed (mean ± S.D.). Statistical analysis was carried out using the paired t-test and data were compared to untreated samples, *** = P<0.001. D. HepG2 cells were incubated −/+ BMP9 (5 ng/ml) for different periods of time in 0.1% FBS media and Id1 levels were analyzed by qRT-PCR and normalized to 18S. Fold changes relative to untreated samples were determined (mean ± S.E.M, n = 3).