Addition of BMP-2 (40 ng/ml) in GM did not significantly change AKT-1 and AKT-2 gene expressions of PASMCs.

<p>No significant reduction or increment of AKT-1 gene expression was found when PASMCs were cultured in GM supplemented with BMP-2 (<b>A</b>). Similarly, no significant change of AKT-2 gene expression was found when PASMCs were cultured in GM supplemented with BMP-2 (<b>B</b>).</p

Growth profile of PASMCs cultured in GM supplemented with BMP-2, GW9662 (PPARγ antagonist) or bpV(HOpic) (PTEN inhibitor).

<p>PPARγ antagonist and PTEN inhibitor were added into respective cell culture medium 1 hour before adding BMP-2 (40 ng/ml). (The number of PASMCs after cultured in GM only was considered as 100%). (*: vs GM only) (Lipo = Lipofetamine −2000).</p

RT-PCR primers and annealing temperature.

<p>RT-PCR primers and annealing temperature.</p

Addition of BMP-2 (40 ng/ml) in GM increased PTEN gene expression of PASMCs.

<p>BMP-2 significantly increased PTEN expression at 8 and 24 hours after treatment. (&: vs any other time point, p<0.05; *: vs 0, 1, and 4 hours, p<0.05).</p

BMP-2 increased caspases 3, 8, and 9 activities.

<p>BMP-2 (40 ng/ml) significantly increased caspases 3 (<b>A</b>), 8 (<b>B</b>), and 9 (<b>C</b>) activities of PASMCs cultured in GM. However, pre-treat PASMCs with GW9662 and bpV(HOpic) reversed this effect (*: vs other samples, p<0.05).</p

Reduced PASMCs proliferation rate as a function of BMP-2 concentration when cultured in GM under hypoxia (A).

<p>PASMCs cultured in GM supplemented with 0–80 ng/ml BMP-2. BMP-2 at the concentrations of 40 and 60 ng/ml significantly inhibited PASMC proliferation as compared with GM without BMP-2. Typical pictures of PASMCs cultured in GM only (<b>B</b>), or GM supplemented with BMP-2 at 40 ng/ml (<b>C</b>). (*: vs 0 ng/ml only, p<0.05) <b>(</b>Magnification = 100×).</p

Smad-4 gene and protein expressions of Lipo-siRNA-Smad-4 transfected PASMCs.

<p>(<b>A</b>) QRT-PCR demonstrated that the lowest Smad-4 gene expression was achieved when 120 nM siRNA-Smad-4 was used to transfect 1×10⁵ PASMCs. (<b>B</b>) Abolishment of Smad-4 protein expression did not affect PTEN protein expression. Qantification of PTEN (<b>C</b>) and pAKT (<b>D</b>) protein expression after normalized to GAPDH (consider as 100%). (<b>E</b>) A non-coding siRNA was used as a negative control to determine the specificity of siRNA-Smad-4 targeting. (Lipo = lipofetamine-2000. *: vs lipo only, p<0.05; &: vs GM only, p<0.05).</p

Typical pictures of EGFP expression from pEGFP lipoplexes transfected PASMC when the ratio between Lipofectamine-2000 and plasmid DNA was at (A) 1∶1, (B) 2∶1, (C) 3∶1 and (D) 4∶1, when 2 µg plasmid DNA was used per 1×10⁵ cells.

<p>(Magnification = 40×).</p

Western blot analysis of PASMCs cultured in GM supplemented with BMP-2 under hypoxia.

<p>(<b>A</b>) A dose dependent effect of BMP-2 on PTEN protein expression when BMP-2 was supplemented up to 80 ng/ml in GM. (<b>B</b>) Quantification of PTEN protein expression when BMP-2 was increased from 0–80 ng/ml after normalized to GAPDH (consider as 100%). (<b>C</b>) Typical pictures of PTEN and pAKT protein expressions as a function of time when BMP-2 was supplemented at 40 ng/ml in GM. Quantification of PTEN (<b>D</b>) and pAKT (<b>E</b>) protein expressions after normalized to GAPDH (consider as 100%). Reduced PTEN protein expression was found when GW9662 was added alone (<b>F</b>) or combined with BMP-2 (<b>G</b>). Reduced PTEN protein expression was also found when bpV (HOpic) was added alone (<b>H</b>) or combined with BMP-2 (<b>I</b>). (*: vs 0 ng/ml BMP-2, p<0.05; &: vs 0 hour, p<0.05).</p

Toxicity of BMP-2 towards PASMCs when PASMCs were cultured in GM supplemented with 0−80 ng/ml BMP-2 under normoxia.

<p>It appears that only at 80 ng/ml concentration of BMP-2 resulted in significantly increased LDH leakage as compared with GM with 0 ng/ml BMP-2. The percentage of LDH leakage was normalized to fresh GM (consider as 0%). (*: vs 0 ng/ml, p<0.05).</p