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. 2010 Apr;24(4):1105–1116. doi: 10.1096/fj.09-141341

Figure 1.

Figure 1.

TGF-β induces EP2 expression in normal and malignant MECs. A) Quiescent NMuMG cells were stimulated with TGF-β1 (5 ng/ml) for 36 h. Detergent-solubilized whole-cell extracts were immunoblotted with antibodies against COX-2, EPs 1-4, and β-actin as indicated. Images represent 3 independent experiments. Immunoblot band densities were quantified using NIH ImageJ. Data are mean ± se pixel density of Cox2 and EPs 1-4 expression relative to control cells (n=3). B) Quiescent 4T1 cells were administered with TGF-β1 (5 ng/ml) or TβRI inhibitor (100 ng/ml; TβRIinhi) for 24 h as indicated. Detergent-solubilized whole-cell extracts were immunoblotted with antibodies against COX-2, EPs 1-4, and β-actin as indicated. Images represent 3 independent experiments. C) Quiescent 4T1 cells were treated with TGF-β1 (5 ng/ml) for 24 h as indicated. Total RNA was isolated and subjected to semiquantitative real-time PCR to monitor the expression of EPs 1-4. Data are mean ± se fold change in EP1-4 gene expression relative to control cells (n=3). D) Quiescent NMuMG or 4T1 cells were treated with TGF-β1 (5 ng/ml) with or without the EP2 antagonist AH6809 (50 μM) as indicated. Detergent-solubilized whole-cell extracts were immunoblotted with antibodies against EP2 or β-actin as shown. Images represent 3 independent experiments. E) Quiescent NMuMG or 4T1 cells were transiently transfected overnight with SBE-luciferase and β-gal cDNAs and subsequently were incubated with antagonists against EP2 (AH6809, 50 μM) or EP4 (GW627368X; 20 μM) for an additional 24 h. Afterward, luciferase and β-gal activities contained in detergent-solubilized whole-cell extracts were measured. Data are mean ± se luciferase activity relative to control cells (n=3). F) Quiescent NMuMG cells were transiently transfected overnight with CRE-luciferase and β-gal cDNAs, and subsequently were incubated with EP2 (butaprost; 10 μΜ) or EP4 (PGE1-alcohol; 1 μΜ) agonists in combination with EP2 (AH6809; 50 μM) or EP4 (GW627368X; 20 μM) antagonists for an additional 24 h as indicated. Afterward, luciferase and β-gal activities contained in detergent-solubilized whole-cell extracts were measured. Data are mean ± se luciferase activity relative to control cells (n=3). G, H) NMuMG (G) or 4T1 cells (H) were transiently transfected overnight with β-gal, together with either SBE- or PAI1-luciferase as indicated. Afterward, cells were stimulated with TGF-β1 (5 ng/ml), forskolin (30 μM), or both agents for 24 h as shown. Afterward, luciferase and β-gal activities contained in detergent-solubilized whole-cell extracts were determined. Data are mean ± se luciferase activity relative to control cells (n=3). *P < 0.05; Student’s t test.