Fig. 5.

Overexpression of the Y705F-STAT3 mutant augments TGF-β1- and NIC+TGF-β1-induced α-SMA expression as well as activation of α-SMA promoter in cultured renal proximal tubule cells. LLC-PK₁ cells were infected with either a negative control (AdNull) or with the Y705F-STAT3 adenovirus as described in materials and methods. Uninfected cells were also included. A: cells were treated with 10 ng/ml TGF-β1 for 3 days, and levels of α-SMA and STAT3 along with actin were determined by Western blotting. B: densitometry results of blots shown in A; n = 3 independent experiments. *P < 0.05 compared with none or as indicated. C: LLC-PK₁ cells were transfected with an α-SMA promoter luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h and then treated with 10 ng/ml TGF-β1 for 24 h. Firefly (α-SMA promoter) and Renilla luciferase activities were determined as described in materials and methods. Values are means ± SD and represent relative luciferase activities (firefly/Renilla) expressed as a percentage of controls; n = 3 independent experiments. *P < 0.05 compared with none or as indicated. D: cells were treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1 for 3 days, and levels of α-SMA and STAT3 along with actin were determined by Western blotting. E: densitometry results of blots shown in D; n = 3 independent experiments. *P < 0.05 compared with none or as indicated. N+T, NIC+TGF-β1. F: LLC-PK₁ cells were transfected with an α-SMA promoter luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h. Cells were treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1. Firefly (α-SMA promoter) and Renilla luciferase activities were determined as described in materials and methods. Values are means ± SD and represent relative luciferase activities (firefly/Renilla) expressed as a percentage of controls; n = 3 independent experiments. *P < 0.05 compared with none or as indicated.