Pwzl neo myr flag pdk1

C2C12 cells were proliferated for 7~10 days to have myoblasts converted to myotubes which contain more than 2 nuclei in a fiber) in the medium with 10% FBS in DMEM media. When C2C12 was reached 70% confluency, the differentiation medium was changed to DMEM with 2% horse serum. Media was replaced every 2~3 days until the myotubes were harvested. The myotubes were infected with empty vectors or Pdk-1 plasmids (Plasmid 20564; pWZL Neo Myr Flag PDK1, Addgene), and then were exposed to hypoxic conditions. The 60 mm dishes were placed in a hypoxia chamber (Billups-Rothenberg Inc.), and internal gases alternated between room air and a hypoxic gas mixture (1% O2, 5% CO2, and 94% N2) every 10 min, eight cycles to mimic intermittent hypoxia conditioning in tissue levels. For treatment, the cells were maintained in the hypoxic gas mixture for 6 h at the final stage of differentiation.

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.