Ciglitazone induces Egr-1 protein expression; silencing of Egr-1 abrogates the effect of ciglitazone on PDK1 promoter activity, protein expression and cell proliferation. A, Cellular proteins were isolated from H1299 cells treated with ciglitazone (20 μM) for the indicated time period (upper panel) or with metformin (5 mM) for 24 h (lower panel). Afterwards, Western blot analyses were performed for detecting Egr-1, p65 and p53 proteins. B, H1299 cells were transfected with control or AMPKα siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h followed by Western blot. C, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) together with a wild type PDK1 promoter construct for 30 h, then cells were exposed to ciglitazone (20 μM) for an additional 24 h. Insert shows the Western blot result for Egr-1 protein. D, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposing the cells to ciglitazone (20 μM) for an additional 24 h, followed by Western blot. E, H1299 cells were transfected with control or Egr-1 siRNA (80 nM) for 30 h before exposure of the cells to ciglitazone (20 μM) for an additional 24 h. Afterwards, the luminescence of viable cells was detected using Cell Titer-Glo Luminescent Cell Viability Assay kit. The bars represent the mean ± SD of at least four independent experiments for each condition. *Indicates significant difference as compared to the control. **Indicates significance of combination treatment as compared with ciglitazone alone (P < 0.05).
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