The washed blots were transferred to freshly made ECL Prime (Pierce, Rockford, IL, USA) and exposed to X-ray film. Cell viability buy AZD1390 assay NSCLC cells (105 cells/well) were transfected with control, PDK1 or PPARα siRNAs for 30 h before exposing the cells to NAC for an additional 48 h in 96-well plates. In parallel experiments, cells
were transfected with control or overexpression PDK1 vector obtained from Addgene . Afterwards, the numbers of viable cells in culture were determined using The CellTiter-Glo Luminescent Cell Viability kit according to the manufacturer’s instructions (Promega, USA). MTT assay Cell viability was analyzed by the MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide] assay. Briefly, cells were seeded in 96-well plates at the density of 1.5 × 103 cells/well and were cultured with NAC for up to 48 h, and then 10 μL of 10 mg/mL MTT solution was added to each well for an additional 4 h according to manufacturer instructions. (Promega, Shanghai, China). After centrifugation, 150 μL dimethyl sulfoxide was
added to the precipitate and the absorbance of the enzyme was measured at 490 nm using an Microplate Reader (Bio-Rad, Hercules, CA, USA). Cell growth rates (average absorbance of each treated group and treated group) were then calculated. All experiments were performed in Selleck Cilengitide Dapagliflozin triplicate samples and repeated
at least three times. Transient transfection assay The original human PDK1 promoter construct was a gift from Dr. Michalik at the University of Lausanne and have been reported previously . The PDK1 promoter construct contains approximately 1500 base pairs of the 5’ flanking region of the human PDK1 gene connected to the pGL2 basic luciferase reporter vector . Briefly, NSCLC cells were seeded at a density of 5 × 105 cells/well in 6-well dishes and grown to 50 –60% confluence. For each well, 2 μg of the above PDK1 plasmid DNA constructs, or overexpression of PDK1(pDONR223-PDPK1) , or p65 vectors (pCMV4 p65)  with 0.2 μg of the internal control phRL-TK Renilla Luciferase Reporter Vector were co-transfected into the cells with the oligofectamine reagent (Invitrogen). In separate experiments, cells were transfected with control or PDK1, PPARα and p53 siRNAs (70 nM each) for 32 h followed by exposed the cells to NAC for an additional 24 h. The preparation of cell extracts and measurement of luciferase activities were carried out using the Dual-Luciferase Reporter Kit according to recommendations by the manufacturer. Changes in firefly luciferase activity were Selleck Smoothened Agonist calculated and plotted after normalization with changes in Renilla luciferase activity within the same sample.