MTS 5 2 2H tetrazolium assay was performed as previously described. Growth inhibition was measured by comparing A490 of drug treated cells to that of untreated controls set selleckbio at 100%. The IC50 value was calculated from sigmoidal dose response curves using Prism 5. 0. Statistical significance of IC50 values between cell lines was evaluated by ANOVA using SAS 9. 3. 1 statistical software. Flow Cytometric/Sub G1 Analysis Cells were seeded at 2. 5 105 cells/well in 6 well plates. Duplicate wells were treated with the indicated concentra tion of drug for 24 h and harvested as previously described. Cells were then analyzed using a Coulter EPICS ELITE flow cytometer equipped with a 15 mW Argon laser. Viable cells were gated on a dot plot dis play of forward scatter versus side scatter to eliminate cell doublets.

Cell cycle populations were quantified using histogram analysis software. Cells with DNA content 1 were considered apoptotic. Western Blotting Following treatment with indicated concentrations of drug for specified time points, Western blot was per AV-951 formed as described previously. Acetylated histones were detected using anti acetyl H3 and anti acetyl H4 rab bit antibodies. Monoclonal anti Poly Polymerase was obtained from Cell Sign aling. Secondary antibodies goat anti mouse HRP or goat anti rabbit HRP were purchased from Santa Cruz Biotechnology. Anti actin was purchased from Sigma and used to control for loading. Microarray Drug Treatments and RNA Isolation HCT116 and HT29 colon cancer cells were seeded at 7 106 cells/10 cm plate and treated with either 50 nM LBH589 or 2 M vorinostat for 24 h at 37 C and 5% CO2.

All treatments were conducted in triplicate and fresh medium was added to untreated control cells. Following the 24 h incubation, cells were harvested and RNA was isolated using the RNeasy Mini Kit according to the manufacturers protocol. RNA was subjected to lithium chloride precipitation www.selleckchem.com/products/MDV3100.html to remove any possible genomic DNA contamination. The integrity of the RNA was analyzed by spectrophotometry and capil lary electrophoresis. Microarray Expression Profiling Microarray expression profiling was performed by the USC/Norris Cancer Center Genomics Core Facility. The RNA was amplified into cRNA and biotinylated by in vitro transcription using the Illumina TotalPrep RNA Amplification Kit according to the manufacturers protocol. Biotinylated cRNAs were purified, fragmented, and subsequently hybridized to an Illumina Human 6 V2 BeadChip. Data normalization and statistical analysis Microarray statistical analysis was performed with the assistance of Asuragen Inc,The background subtraction, expression summary, normalization, and log base 2 transformation of gene signals were carried out using Quantile Normalization.