Plasmid construction and mutagenesis fragments of the human IBP gene were amplified from the genomic DNA of MCF 7 cells by PCR using KOD poly merase. These amplified fragments were inserted into the KpnI and HindIII restriction sites of the pGL3 basic vector. The wild type p53 ex pression plasmid, pCMV p53, and the p53 mutant plasmid, pCMV p53R175H, sellekchem were kindly provided by Dr. Vogelstein. TaKaRa MutanBEST kit was used to introduce the p53 binding site into the IBP promoter deletion mutant. The following mutagenic primers. The pEGFP C1 IBP expres sion plasmid was a gift from Dr. Alessandra B. Pernis. All of the constructs were confirmed by DNA sequencing. Adenovirus infection Inhibitors,Modulators,Libraries and cell treatment Adenovirus p53 was purchased from Shenzhen SiBiono GeneTech Co.

Ad GFP was purchased from Inhibitors,Modulators,Libraries Shanghai Sunbio Medical Biotechnology Co. The cells were treated with different concentrations of doxo rubicin for 8 h, Nutlin 3 for 24 h and pifithrin for 24 h. The cisplatin concentrations and experimental details are described in the text and figure legends. The cells were treated with Ly294002 or wortmannin for 24 h. RNA interference To knockdown IBP expression, double stranded Inhibitors,Modulators,Libraries DNA oligonucleotides were subcloned into pcDNA 6. 2 GWEmGFPmiR using the BLOCK iT Pol II miR RNAi Expression Vector Kit. The RNAi plasmid or control plas mid, which contained a non specific sequence, was transfected into MCF 7 cells. Lipofectamine 2000 was used as the transfection reagent. The growth medium was supplemented with blasti cidin, which was used to se lect for blasticidin resistant transfectants.

For the p53 knockdown, double stranded DNA oligonucleo tides were subcloned into pMagic 1. 1 and packaged into lentivirus particles. One day after infection, the cell growth medium was supplemented with puromycin Inhibitors,Modulators,Libraries to select stable transfectants. Luciferase reporter assays Luciferase reporter assays were performed using the Dual LuciferaseW Reporter Assay System. Cells were seeded in 24 Inhibitors,Modulators,Libraries well plates and transfected together with a promoter reporter gene vector and the pRL TK Renilla luciferase vector. After 48 h of transfection, the cells were harvested and ana lysed according to the manufacturers instructions. The luciferase activities were normalised to the Renilla luci ferase activity of the internal control. Western blotting Cell lysates were prepared in RIPA buffer.

Whole cell lysates were separated on a 10% SDS PAGE gel and transferred onto polyvinylidene difluoride Perifosine clinical trial membranes. The membranes were blocked for 1 h at 37 C in 5% non fat milkTBST and were then incu bated with primary antibodies overnight at 4 C. Antibodies against IBP, p53 phospho p53, Bcl 2, Bax, phospho AKT, AKT,phospho MDM2, MDM2 and GAPDH were used. The membrane was then rinsed in TBST and incubated with various secondary antibodies for 2 h at 25 C. Immunoreactive bands were visualised with a chemiluminescent HRP substrate.