Figure 4.

Ciglitazone decreased PDK1 promoter activity. A, The human PDK1 wild type reporter construct schematics are presented. These regions contain several transcription factor binding sites including PPRE, Egr-1, p53 and NF-κB. B, H1299 and H1650 lung cancer cells (1 × 10⁵ cells) were transfected with a wild type human PDK1 promoter reporter construct ligated to luciferase reporter gene and an internal control Renilla Luciferase Reporter Vector for 24 h. Afterward, cells were treated with ciglitazone for an additional 24 h. C, H1299 cells were transfected with control or PPARγ siRNAs (80 nM) together with a wild type ILK promoter construct for 30 h, then cells were exposed to ciglitazone for an additional 24 h. Insert on the top shows Western blot result for PPARγ protein. GAPDH served as internal control for normalization purposes. D, H1299 cells (1 × 10⁵ cells) were transfected with a wild type human PDK1 promoter reporter construct ligated to luciferase reporter gene and an internal control Renilla Luciferase Reporter Vector as described in Materials and Methods for 24 h. Afterwards, cells were treated with SP600125 (10 μM) for 1 h before exposure of the cells to ciglitazone for an additional 24 h. The ratio of firefly luciferase to renilla luciferase activity was quantified as described in Material and Methods. The bars represent the mean ± SD of at least four independent experiments for each condition. *Indicates significant increase of activity as compared to controls. **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).