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Cell cycle analysis. Following treatment, CP868596 were collected by trypsinization and centrifugation, then washed with PBS, and fixed with 70% ethanol. Cells were labeled with propidium iodide (PI) solution (0.05 mg/ml PI, 2 mg/ml RNase A, 0.01% Triton X-100 in PBS) and incubated for 30 min at room temperature in darkness. Cell cycle (DNA content) was analyzed using an Epics XL-MCL Flow Cytometer (Beckman Coulter, Fullerton, CA).
Determination of apoptosis by flow cytometry. Following treatments, attached and detached cells were harvested, washed with cold PBS, resuspended in 1× binding buffer (0.1 M HEPES/NaOH, pH 7.4, 1.4 M NaCl, 25 mM CaCl2), stained with Annexin V-FITC/PI staining kit (BD Bio-sciences, San Jose, CA) and incubated for 15 min at room temperature in darkness. Cells were analyzed using an Epics XL-MCL Flow Cytometer (Beckman Coulter).
Protein extraction and Western blot analysis. Western blotting was used for analyzing specific proteins. Briefly, total protein samples from cells were denatured, and loaded onto the SDS–polyacrylamide gradient (4–20%) gels (Bio-Rad, Hercules, CA), resolved by electrophoresis, and electroblotted to membranes. The blots were incubated with a primary IgG antibody followed by incubation with an alkaline horseradish peroxidase (HRP)-conjugated secondary IgG antibody. Specific protein bands on the blots were detected by HRP/H2O2 catalyzed oxidation of luminol in alkaline condition using enhanced chemiluminescence (ECL) system (GE Healthcare Bio-Sciences, Piscataway, NJ) followed by autoradiography. Autoradiograms were scanned on a UMAX PowerLook Scanner (UMAX, Fremont, CA) using Photoshop software (Adobe Systems, Seattle, WA), and OD of each band was determined using NIH Image software.