Control nonspecific sirna oligonucleotides

The EP4, PDK1, c-Jun siRNA and control nonspecific siRNA oligonucleotides were purchased from Santa Cruz Biotechnology. pWZL-Neo-Myr- Flag PDK1 and pWZL-Neo-Myr-Flag-DEST were purchased from Addgene Inc. The pGME4 c-Jun vector and pGME4 were provided by Dr. Tom Curran (Children’s Hospital of Philadelphia, University of Pennsyvania, USA). For the transfection procedure, cells were grown to 60 % confluence, and EP4, c-Jun and PDK1 siRNAs,control siRNA,and expression vector were transfected using the lipofectamine 2000 reagent according to the manufacturer’s instructions. Briefly, the lipofectamine reagent was incubated with serum-free medium for 5 min. Subsequently, a mixture of respective siRNA was added. After incubation for 20 min at room temperature, the mixture was diluted with medium and added to each well. The final concentration of siRNAs in each well was 100 nmol/L. After culturing for 30 h, cells were washed and resuspended in new culture medium in the presence or absence of dmPGE₂ for Western blot and cell growth and gel mobility shift assays.

The siRNA human PDPK1 (EHU071261) was ordered from Sigma (Shanghai, China). The AMPKα (Cat No.sc-45312), Egr-1 siRNA (Cat No. sc-105070), PPARγ siRNA (Cat No. sc-29455), and control nonspecific siRNA oligonucleotides (Cat No. sc-37007) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). For the transfection procedure, cells were grown to 60% confluence, and PDK1, Egr-1, and PPARγ and control siRNAs were transfected using the oligofectamine reagent (Invitrogen, Shanghai, China) according to the manufacturer’s instructions. Briefly, Lipofectamine was incubated with serum–free medium for 10 min., mixed with siRNA (80 nM), incubated for 20 min at room temperature before the mixture was diluted with medium and added to cells. After culturing for 30 h, cells were washed, resuspended in new culture media in the presence or absence of ciglitazone for an additional 24 h for Western Blot, cell growth, luciferase report assays and other experiments.