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 blog post 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 to select stable transfectants. Luciferase reporter assays Luciferase reporter assays were performed using the Dual LuciferaseW Reporter Assay System. Cells were seeded in 24 well plates and transfected together with a promoter reporter gene vector and the pRL TK Renilla luciferase vector. After 48 h of transfection, Inhibitors,Modulators,Libraries 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.

Inhibitors,Modulators,Libraries 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 membranes. The membranes were blocked for 1 h at 37 C in 5% non fat milk/TBST and were then incu bated with primary antibodies overnight at 4 C. Antibodies against IBP 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. Quantitative RT PCR Total RNA was isolated using TRIzol, and 1 ug of isolated RNA was reverse transcribed to generate cDNAs. Amplification was performed by using SYBR Premix Ex Taq II. GAPDH mRNA levels were determined as an internal control.

Electrophoretic mobility shift assays Nuclear extracts were prepared in hypertonic Inhibitors,Modulators,Libraries buffer. were la belled with 32P ATP using T4 polynucleotide kinase. The nuclear extracts were incubated with the probe for 30 min at 30 C. The protein DNA complexes were resolved using non denaturing PAGE and were detected by autoradiography. For the cold probe compe tition assay, unlabelled probe was added to the nuclear protein extracts one hour before the detection was per formed. In the supershift assay, 1 ul of an anti p53 anti body was incubated with the nuclear extracts for 1 h at room temperature prior to Inhibitors,Modulators,Libraries the addition of the radiolabeled probe and the implementa tion of PAGE. Cell survival assays A cell survival assay was performed in triplicate with a Cell Counting Kit 8.

The cells were seeded in 96 well plates Inhibitors,Modulators,Libraries at 5 103 cells/well 24 h before the cisplatin treatment. The culture medium was then replaced with fresh selleck catalog medium that con tained different concentrations of cisplatin, which ranged from 0 to 32 ug/ml, and the cells were cultured in this medium for 24 h. Following the incubation, 10 ul of CCK 8 solution was added to each well, and after 1 h, the absorbance value of each well was read at 450 nm. The cell growth rate was calculated as the ratio of the absorbance of the experimental well to that of the blank well.