Plates were then washed selleck kinase inhibitor and Inhibitors,Modulators,Libraries nuclei stained with 10 uM Hoechst 33342 in a fixation solution with 3. 7% formaldehyde. To study cell cycle arrest, HCT116 cells were incubated for 24 h with VLX40. Cells were stained using Cell Cycle Kit I reagents for DNA content and phospho histone H3 staining according to the manufacturers instructions. Primary antibodies specific for phospho histone H3, secondary antibodies DyLight 549 Conjugated Goat anti Rabbit IgG and DAPI dye were used. Processed plates were loaded in the ArrayScan and analyzed. Images were acquired for each fluorescence channel, using suitable filters with 10X or 20X objective and in each well at least 1000 cells were analyzed. Quan tification of apoptosis was performed by measuring caspase 3 activation and nuclear fragmentation, wheras quantification of cell cycle arrest was obtained by nuclear DNA content and phospho histone H3.

Flow cytometry analysis of cell cycle and apoptosis Cells were seeded in 24 well plates 24 h Inhibitors,Modulators,Libraries prior to treatment with different concentrations of VLX40 for 6, 16, 24 and 48 hours. Upon Inhibitors,Modulators,Libraries drug exposure, cells were washed with PBS and stained with Annexin Inhibitors,Modulators,Libraries V FITC according to the instructions of the vendor. Cell cycle analysis was performed by labeling digitonin permeabilized cells with 5 ug/ml propidium iodide. Flow cytometry analysis was performed using a BD LSR II flow cytometer. Phase contrast microscopy Time lapse phase contrast microscopy was performed using an automated IncuCyte phase contrast microscope. MCF 7 cells were plated on 24 well ImageLock plates and immediately placed into the IncuCyte imaging system.

The Inhibitors,Modulators,Libraries chamber is designed to fit into a standard, humidified incubator in an atmosphere of 5% CO2, and a moving objective allows the cell culture to be stationary while images are captured at different positions from well to well. Images were collected at 1 h intervals starting 30 min after placing the plate in the IncuCyte chamber and cells were left to attach for 24 h when drug treatment was performed. Cell density was calculated using the IncuCyte software. Microarray analysis RNA from cell cultures was isolated using RNeasy Mini Kit from Qiagen and immediately stored at 70 C until further use. RNA purity and quality was measured using an ND 1000 spectrophotometer and Bioanalyzer 2100, respectively. Starting from 2 ug of total RNA, gene expression analysis was performed using Genome U133 Plus 2.

0 Arrays according to the GeneChip Expression Analysis Technical Manual. Raw data was nor malized using MAS5. Connectivity Map build 02 contains genome wide expression data for 1,309 compounds. The original protocol using MCF 7 breast cancer cells as described by Lamb et al. was used. Briefly, cells were selleck chemical seeded in a 6 well plate at a density of 0. 4 106 cells per well. Cells were left to attach for 24 h, followed by exposure to either VLX40 at a final concentration of 10 uM, or to vehicle control.