Figure 2

NAC induces protein expression of PPARα; Blockade of PPARα abrogates the inhibitory effect of NAC on PDK1 expression and cell growth. A-B, Cellular protein was isolated from A549 cells that were cultured with increased concentrations of NAC for 24 h (A) or cultured with NAC (5 mM) for the indicated time (B), followed by Western blot analysis with antibodies against PPARα. The bar graphs represent the mean ± SD of PPARα/GAPDH of three independent experiments. *indicates significant difference from untreated control. C, Cellular protein was isolated from NSCLC cell lines that were cultured with NAC for 24 h followed by Western blot analysis with antibodies against PPARα protein. GAPDH used as loading control. CTR, indicates untreated cells. D, A549 cells were treated with GW6470 (20 μM) for 2 h before exposure of the cells to NAC (5 mM), Fenofibrate (10 μM) for an additional 24 h. Afterwards, Western blot analysis was performed to detect PDK1 protein. E, Cellular protein was isolated from A549 cells transfected with control or PPARα siRNA (100 nM each) for 30 h before exposure of the cells to NAC (5 mM) for an additional 24 h. Afterwards, Western blot analysis was performed to measure PPARα and PDK1 proteins. The bar graphs represent the mean ± SD of PDK1/GAPDH of three independent experiments. *indicates significant difference from untreated control. **indicates significance of combination treatment as compared with NAC alone (P < 0.05). F, A549 and H1650 cells were treated with GW6470 (20 μM) for 2 h before exposure of the cells to NAC (5 mM), Fenofibrate (10 μM) for an additional 48 h. Afterwards, the luminescence of viable cells was detected using Cell Viability Assay Kit. All data were depicted as mean ± SD. *indicates significant difference as compared to the untreated group (CTR).