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Figure 5 | Respiratory Research

Figure 5

From: Differential effects of formoterol on thrombin- and PDGF-induced proliferation of human pulmonary arterial vascular smooth muscle cells

Figure 5

Formoterol does not affect mTORC1 and mTORC2 activation by chronic hypoxia, thrombin and PDGF. A: Human PAVSM cells serum-deprived for 48 h were treated with 1 U/ml thrombin (thr), 10 ng/ml PDGF, or diluent (dil) for 30 min, and then immunoblot analysis with anti-phospho S6, anti-total S6, anti-phospho S473-Akt, and anti-total Akt was performed. Images are representative of three independent experiments. B: Chronic hypoxia promotes S6, S6K1, and S473-Akt phosphorylation in human PAVSM cells. Human PAVSM cells serum-deprived for 48 h maintained under chronic hypoxia (hyp) or normoxia (norm) were subjected to immunoblot analysis with specific antibodies to detect indicated proteins. Images are representative of three independent experiments. C, D: Formoterol has no effect on S6 and S473-Akt phosphorylation. C: Serum-deprived cells maintained under hypoxia or normoxia were incubated for 30 min with 10 μM (R,R)-, (S,S), or racemic formoterol, treated with different concentrations of thrombin and PDGF, or diluent, and then immunoblot analysis with anti-phospho-S6 and anti-total S6 antibodies was performed. Representative images (top) and statistical analysis (bottom) of three independent experiments. Data represent P/total S6 ratio (n = 3 for each experimental condition). P/total S6 ratio for diluent-treated normoxia-exposed cells was taken as one fold). Data are means ± SE by ANOVA (Bonferroni-Dunn). *p < 0.001 for thrombin vs. diluent, thrombin + (R,R), (S,S) and racemic formoterol vs. (R,R), (S,S) and racemic formoterol, respectively. D: Immunoblot analysis of serum-deprived normoxia-maintained cells treated as described above was performed to detect P-S473-Akt and total Akt levels. Representative images (top) and data analysis (bottom) from three separate experiments. Data represent P-S473/total Akt ratio (n = 3 for each experimental condition) normalized by P/total Akt for diluent-treated cells (taken as one fold). Data are means ± SE by ANOVA (Bonferroni-Dunn). *p < 0.001 for PDGF vs. diluent, PDGF + (R,R) formoterol vs. (R,R) formoterol, PDGF + (S,S) formoterol vs. (S,S) formoterol, and PDGF + racemic formoterol vs. racemic formoterol.

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