Briefly, 15,000 cells were plated in 96 well AZD9291 molecular weight plates and treated with CRF At the end of the incuba tion period MTT was added at a concentration of 0. 5 mg ml and incubated for 3 hours. Cells were then lysed by adding 0. 04 N HCl in isopropanol and absorbance was measured at 620 nm in an ELISA plate reader. Wound healing assay Cells were cultured in 60 mm plates until the surface was completely covered. A small area was then disrupted and a group of cells was destroyed or displaced by scratching a line through the layer with a tip. The culture medium was replaced with serum free medium and cells were stim ulated with 10 8 M CRF. The open gap was then inspected microscopically over time as the Inhibitors,Modulators,Libraries cells moved in and filled the damaged area.

Images were cap tured at the beginning and at regular time points during cell migration and the cell migration was quantified by measuring the distance with the program Image between two certain points on either side of the gap. For proper statistical evaluation, Inhibitors,Modulators,Libraries at least three measurements at different points were performed at each image. Cell Invasion Assay The assay was performed in a 96 well invasion plate based on the Boyden chamber principle. The bottom of each well contained an 8m pore size polycarbonate mem brane coated with a thin layer of Extracellular Matrix through which invasive cells migrate to the bot tom of the membrane. Invaded cells were dissociated, lysed and quantified by fluorometric analysis using SYBR green, according to the manufacturers instructions. Evaluation of actin reorganization by Confocal Laser Scanning Microscopy Cells were cultured in 8 well chambers slides.

The next day the culture medium was replaced with serum free medium and cells were stimulated with 10 8 M CRF for 1, 3 and 6 hours. At the end of each exper iment, cells were harvested, Inhibitors,Modulators,Libraries transferred to tubes, washed Inhibitors,Modulators,Libraries with PBS and permeabilized by exposure to 3. 7% formal dehyde Inhibitors,Modulators,Libraries for 10 minutes. Cells were then incubated with acetone for 4 minutes at room temperature, washed with PBS and incubated with 1. 5% FCS. Finally, rhodamine phalloidin was added to the cells at 1 100 dilution in PBS FCS 1. 5% for 30 min in the dark. Subsequently, cells were washed with PBS, analyzed with a confocal laser scanning module and images were assessed with the respective software.

Measurement of monomeric and polymeric actin by Triton X 100 fractionation The Triton X 100 soluble G actin and insoluble F actin containing fractions of cells exposed to CRF at 10 8 M in serum free medium sellekchem for 3 and 6 hours were prepared as previously described. The quantification of actin was performed by reference to a standard curve, prepared from muscle actin. The G and total actin contents were related to the total protein content. Protein concentra tions were measured with a commercially available kit. A decrease of the triton soluble to total actin ratio is indicative of actin polymerization.