Fig 1.
Ouabain down-regulates TGFβR2 mRNA and protein levels in human lung fibroblasts (HLF).
Serum-starved HLF were treated with 100 nM ouabain for 3, 6, and 24 hours. Cells were analyzed by real-time qPCR for TGFβR2 mRNA levels (A) and by Western blotting for TGFβR2 protein levels (B). Shown are the representative images and the quantitative analysis of ECL (mean±SD) from at least three independent experiments (RLU, relative light units). **p < 0.005, ***p < 0.0005. C. HLF isolated and cultured from IPF and non-IPF (NL) lungs were treated with or without 100 nM ouabain. Cells lysates were analyzed for TGFβR2 or tubulin protein expression by Western blotting.
Fig 2.
Inhibition of Na+/K+ ATPase by potassium free media results in the down-regulation of TGFβR2 mRNA and protein levels in human lung fibroblasts (HLF).
A. HLFs were placed in potassium free media for 3, 6, and 24 hours, and then analyzed for TGFβR2 mRNA levels via real time qPCR. B. HLFs were treated with media containing either 5 mM KCl or 0 mM KCl for 24 or 48 hours. Cell extracts were examined by Western blotting for TGFβR2 expression or tubulin. Shown are the representative images and the quantitative analysis of ECL (mean±SD) from at least three independent experiments (RLU, relative light units). **p < 0.005, ***p < 0.0005.
Fig 3.
Inhibition of the Na,K-ATPase by potassium free media or by ouabain blocks TGFβ1-induced SMAD2 phosphorylation.
HLF were pretreated with ouabain at 10, 30,100 nM (A) or placed in media containing 5 mM KCl or 0 mM KCl (B) for 24 hours. Cells were then stimulated with 1ng/ml of TGFβ1 for 1 hour. Cell lysates were analyzed by Western blotting with desired antibodies as indicated. Shown are the representative images and the quantitative analysis of ECL (mean±SD) from at least three independent experiments (RLU, relative light units). *, p < 0.05.
Fig 4.
Inhibition of the Na,K-ATPase by potassium free media and ouabain blocks TGFβ1-induced myofibroblast differentiation.
HLF were treated with increasing doses of ouabain (A) or placed in media containing 5 mM KCl or 0 mM KCl (B) and stimulated with or without 1 ng/ml of TGFβ1 for 24 hours. Cell lysates were then analyzed by Western blotting with desired antibodies as indicated. Shown are the representative images and the quantitative analysis of ECL (mean±SD) from at least three independent experiments (RLU, relative light units). *p < 0.005, **p < 0.0005.
Fig 5.
Downregulation of TGFβR2 by ouabain is accompanied by inhibition of TGFβ1-induced SMAD2 phosphorylation and of the expression of mesenchymal markers in A549 cells.
Serum starved A549 cells were pretreated with increasing doses of ouabain for 24 hours followed by stimulation with 10ng/ml of TGFβ1 for 1 hour (A) or simultaneously treated with ouabain and TGFβ1 for 48 hours (B). Cell lysates were then analyzed by Western blotting with desired antibodies as indicated. Shown are the representative images and the quantitative analysis of ECL (mean±SD) from at least three independent experiments (RLU, relative light units). *, p < 0.05, **p < 0.005, ***p < 0.0005.