Fig. 6.

Overexpression of the U-STAT3-mimetic Y705F-STAT3 mutant augments NIC+TGF-β1-induced activation of MCP-1 promoter as well as expression of profibrotic genes in cultured renal proximal tubule cells. A: LLC-PK₁ cells were transfected with an MCP-1 promoter luciferase reporter plasmid along with a Renilla luciferase plasmid for 24 h followed by infection with either an empty adenovirus (AdNull) or with the Y705F-STAT3 (ad705F) adenovirus as described in materials and methods. Cells were then treated with 200 μM NIC for 24 h followed by treatment with 10 ng/ml TGF-β1 for 24 h. Firefly (MCP-1) 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. B: LLC-PK₁ cells were transfected with adY705F and then treated with NIC and TGF-β1 as described in materials and methods. Expression of vimentin, fibronectin, and E-cadherin along with actin was determined by Western blotting. Results shown are representatives of 3 independent experiments. C: densitometry of results in B; n = 3. YF, adY705F-STAT3. *P < 0.05 compared with none. #P < 0.05 compared with the corresponding N+T values.