2,5-Dimethoxy-4,5-iodoamphetamine hydrochloride (DOI), ketanserin, ritanserin and demecolcine were purchased from Sigma Aldrich. Stock solutions were prepared in H2O or dimethyl sulfoxide (DMSO) as appropriate and stored at −20°C. Depending on the used agonist/antagonist, 0.1%

DMSO Tyrosine Kinase Inhibitor Library was added to the cell culture media in control groups. After 48-hour serum deprivation, cells were pulsed with 0.1 μCi/well methyl-3H thymidine (100 Ci/mmol, Amersham Pharmacia Biotech), and incubated for 48 hours. Plates were harvested by rinsing in cold phosphate-buffered saline (PBS) followed by a 60-minute incubation in cold 10% trichloroacetic acid (TCA). Wells were washed with 10% TCA and 500 μL of 1N NaOH was added. Then 250 μL of this suspension was neutralized with 250 μL 1N HCl and added to 5 mL scintillation fluid. Measurement was performed in a liquid scintillation counter (Kontron Instruments). 5-Bromo-2′-deoxy-uridine ABT-263 (BrdU) was added after 48-hour serum withdrawal to a final concentration of 10 μM. Labeling and detection of the cell were performed with BrdU-immunofluorescence

following the manufacturer’s instructions (Roche Applied Science). Viable cells were distinguished with the fluorescent dyes Calcein AM and Ethidium homodimer 1 (Live/Dead, Viability/Cytotoxicity Kit, Molecular Probes, L-3224). Stainings were performed according to the manufacturer’s instructions. The number of viable cells was quantified by the addition of 25 μL of a 0.5% tetrazolium salt solution [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT); Sigma Aldrich]. After 45 minutes of incubation, the formation of the formazan product was monitored by measuring absorbance at 570 nm after solubilization

in acidic isopropranol (5% formic acid in isopropranol). Values were calculated as the percentage of untreated controls. Cytotoxicity was assessed using CytoTox-Fluor-Assay (Promega, G9260) following the manufacturer’s instruction. The test measures the relative number of Akt inhibitor dead cells in cell populations and uses a fluorogenic peptide substrate (bis-alanyl-alanyl-phenylalanyl-rhodamine 110; bis-AAF-R110) to measure “dead-cell protease activity,” which has been released from cells that have lost membrane integrity. DNA fragmentation was visualized with fluorescein-dUTP [TUNEL (TdT-mediated dUTP-X nick end labeling)] using a Cell Death Detection Kit (Roche Applied Science). Caspase-3 activity was measured using Caspase-Glo 3/7 Assay (Promega) as well as with a pan-caspase inhibitor (Z-DEVD-FMK) and caspase-3 substrate (Ac-DEVD-AFC) according to the manufacturer’s instruction. Preparation of cell extracts, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) blotting, and secondary staining were performed according to standard protocols.