Human cell culture, treatments, imaging and cell cycle analysis The HeLa cell and MCF 7 cell lines were purchased from ATCC. http://www.selleckchem.com/products/Pazopanib-Hydrochloride.html HeLa cells were grown in MEM supple mented with 10% foetal calf serum, 2 mM L glu tamine, penicillin, streptomycin and non essential amino acids, at 37 C in 5% CO2. Media, serum and reagents for tissue culture were purchased from GIBCO . MCF 7 cells were grown in MEM supplemented with 10% FCS, non essential amino acids, 1 mg/100 ml insulin, 8. 4 mg/100 ml NaHCO3 at 37 C in 5% CO2. HT/HCA Image analysis typically 5000 HeLa and 10000 MCF 7 cells were seeded in 96 well Greiner Bio One plates and left to attach overnight in their respec tive media. The indicated amounts of FTI 277 or the vehicle were added and the cells were grown for a further 48 hours.

Cells were then washed in 1�� phosphate buffered saline and fixed for 10 min with 4% paraformaldehyde in 1 PBS, and permeabi lized for 30 min in blocking buffer. Nuclei were stained with Hoechst, washed three times and inspected using a 20�� objective of the ScanR imaging platform . Sixteen images were acquired randomly per well. For cell cycle analysis at least three wells per con dition were considered for statistical analysis of the HeLa and MCF 7 cells. For all the others five wells were considered. Determination of mitotic cell number fixed cells were incubated for 75 min with a PhosphoH3 pSer10 diluted 1 100 in blocking buffer, washed three times in 1 PBS, and further incubated for 30 min with anti mouse Alexa488 conjugates diluted 1 500 in blocking buffer, washed three times and inspected using the ScanR microscope.

For determination of Pak1/2/3 localization, Cilengitide an anti Pak C19 antibody and anti rabbit Alexa546 were used. For determination of Aurora A localization, an anti IAK1 and anti mouse Alexa488 conju gates were used. HeLa cells were treated for 48 hours with 2 uM of FTI 277 or with the vehicle, and then fixed using the procedure indicated above. To determine the phosphorylation of ribosomal pro tein S6, an Alexa555 conjugated anti PhosphoS6 anti body was used. Typically 4000 HeLa and 8000 MCF 7 cells were seeded in five wells per experimental condition. Cells were left to attach over night, and treated with 5 uM of FTI 277 or with the vehicle for 24 hours in starvation medium. Cells were fixed, washed and probed with Hoechst and processed for ScanR acquisition as described above.

Quantification and statistical analysis were per formed with the in built ScanR analysis software. Introduction Although significant advances have been made in selleck chem the treat ment of acute lymphoblastic leukemia especially in children, only 30 40% of adults have a long term survival. A major subclass of ALL with a specially poor progno sis in both adults and children is that of Philadelphia chromosome positive ALL. The Ph chromosome is generated by a reciprocal t translocation. It is found in around 30% of cases of adult ALL and is the hallmark of chronic myeloid leukemia.