Pwzl neo myr flag dest

The retroviral vector encoding CKB (pWZL-Neo-Myr-Flag-CKB) and the empty vector (pWZL-Neo-Myr-Flag-DEST) were purchased from Addgene (Cambridge, MA). 293 T cells were co-transfected with pWZL-Neo-Myr-Flag-DEST or pWZL-Neo-Myr-Flag-CKB along with gag-pol and VSV-G (8:4:3 ratio). After 48 h, media containing viral soup was concentrated, treated with 8 μg/ml polybrene (Sigma-Aldrich, St. Louis, MO), and added to the MDA-MB-231 cells. After 24 h, the media was replaced with fresh media containing 800 μg/ml neomycin (Sigma-Aldrich) to select infected cells.

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.

In order to explore the critical function of AKT or PLK1 in 15d-PGJ2-induced cytotoxicity, U2OS cells (7×10⁵) were transfected with either 8 μg of myc-tagged myristoylated Akt expression vector (pCDEST-myrAkt) or pWZL NeoMyrFlag PLK1 expression vector or their corresponding empty vector (pCS2+ or pWZL-Neo-Myr-Flag-DEST) (all purchased from Addgene, Cambridge, MA, USA) using LipofectAMINE (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's procedure. After transfection, cells were cultured in 10% FBS–supplemented IMDM for 24 h and then subjected to 0.1% DMSO or 15d-PGJ2 treatment.