Western blot analysis www.selleckchem.com/products/kpt-330.html Protein concentrations were determined by the Bio Rad protein assay. Equal amounts of protein from whole cell lysates were solubilized in 2x SDS sample buffer and separated on 10% SDS polyacrylamide gels. Membranes were incubated with antibodies against PDK1, PPARg phosphor AMPK phosphor p SAPK JNK and total AMPK and SAPK JNK, p53, p65 and Egr 1. The membranes were washed and in cubated with incubation with a secondary goat antibody raised against rabbit IgG conjugated to horseradish peroxidase. The membranes were washed again and transferred to freshly made ECL solution for 1 min, and exposed to X ray film. MTT cell viability assay Cell viability was measured using the 3 2, 5 diphenyltetrazolium bromide assay. Briefly, NSCLC cells were counted and seeded into a 96 well microtiterplate.

The cells were treated with increasing concentrations Inhibitors,Modulators,Libraries of ciglitazone for up to 72 h. After incubation, 10 uL MTT solution was added to each well and incubated at 37 C for an additional 4 h. Supernatant was removed, then 150 uL DMSO was added to each well and oscillated for 10 min. Absorbance at 490 nm was determined through the use of ELISA reader. Each experiment was repeated at least three times. Cell viability was calculated as 100%. CellTiter Glo luminescent cell viability assay Human lung carcinoma cells were treated with com pound C for 2 h or were transfected with control or Egr 1 siRNA or PDK1 expression vectors for 24 h before exposure of the cells to ciglitazone for an add itional 24 h in 96 well plates in DMEM media with Inhibitors,Modulators,Libraries 0. 5% FBS.

Afterwards, cell viability was measured using the CellTiter Glo Luminescent Cell Viability Assay kit according to the instructions of the manufacturer. Detection of caspase 3 7 activity Enzymatic activity of caspase 3 7 was measured using the Caspase Glo 3 7 Assay kit according to the manufacturers instruction. Briefly, NSCLC cells were seeded in 96 well plates and treated with Inhibitors,Modulators,Libraries or without 20 uM of ciglitazone for 48 h. Afterwards, the cells were lysed and incubated with 100 uL of Apo ONE Caspase 3 7 reagent. After 1 h incubation in the dark at RT, the fluor escence of each well was measured at 485 520 nm by reading in an Epoch microplate reader. Treatment with AMPK, PDK1, Egr 1 and PPAR small interfering RNA The siRNA human PDPK1 was ordered from Sigma.

The AMPK, Egr 1 siRNA, PPAR siRNA, and control nonspecific siRNA oligonucleotides were purchased from Santa Cruz Biotechnology. For the transfection procedure, cells were grown to 60% conflu ence, and PDK1, Egr 1, and PPAR and control siRNAs Inhibitors,Modulators,Libraries were transfected using the oligofectamine reagent according to the manufacturers instructions. Briefly, Lipofectamine was incubated with serum free medium for 10 min.mixed with siRNA, incubated for 20 min at room temperature before the mixture was Inhibitors,Modulators,Libraries diluted Calcitriol mechanism with medium and added to cells.