5 × 103 (CH1), 2.5 × 103 (SW480), 4.0 × 103 (A549), 6.0 × 103 (N87 and T47D) and 1.0 × 104 (LNCaP) viable cells per well. Cells were allowed for 24 h to settle find more and resume exponential growth in drug-free MEM, followed by the addition of dilutions of the test compounds in aliquots of 100 μL/well in the same medium (eventually containing not more than 0.5% DMSO). After continuous exposure for 96 h, the medium was replaced by 100 μL/well RPMI 1640 medium plus 20 μL/well solution of MTT in phosphate-buffered
saline (5 mg/mL) (all purchased from Sigma-Aldrich). After incubation for 4 h, medium/MTT mixtures were removed, and the formazan precipitate formed by viable cells was dissolved in DMSO (150 μL/well). Optical densities at 550 nm were measured with a microplate reader (Tecan Spectra Classic), using a reference wavelength of 690 nm to correct for unspecific absorption. The quantity of viable cells was expressed as percentage of untreated controls, and 50% inhibitory concentrations (IC50)
were calculated from concentration–effect curves by interpolation. Evaluation is based on at least three independent experiments, each comprising three replicates per concentration level. The activities of recombinant Cdk2/cyclin E expressed in and isolated from Sf21 insect cells were determined by a radioassay  with minor modifications, using histone H1 as the substrate for phosphorylation. Briefly, MOPS-buffered assay mixtures containing the test compound (and a maximum of 1% DMSO), the kinase/cyclin complex, histone H1 and 0.4 μCi (γ-32P)ATP per sample were Androgen Receptor antagonist incubated for 10 min at 30 °C. Aliquots of the solution were spotted onto phosphocellulose squares, which had been washed 3 times with 0.75% phosphoric acid followed by acetone. The dried squares were measured in scintillation vials
by beta counting (Perkin Elmer Tri-Carb 2800TR; software: Quanta Smart). Results were obtained Loperamide in duplicates in at least two independent experiments. The impact of the compounds on the cell cycle was studied by flow-cytometric analysis of DNA contents of cells stained with propidium iodide. Briefly, 1 million A549 cells were seeded into Petri dishes and allowed to recover for 24 h. Cells were then exposed for 24 h to the test compounds dissolved in a medium containing a maximum of 0.5% DMSO. Control and treated cells were collected, washed with PBS (phosphate-buffered saline), fixed in 70% ice-cold ethanol, and stored at − 20 °C. To determine cell cycle distribution, cells were transferred in physiological saline (0.9% w/v aqueous NaCl solution) into PBS, incubated with 10 μg/ml RNAse A for 30 min at 37 °C, followed by treatment with 5 μg/ml propidium iodide (PI) for 30 min. Fluorescence of 10 000 cells was measured with a FACS Calibur instrument (Becton Dickinson). The resulting DNA histograms were quantified by using the Cell Quest Pro software (Becton Dickinson).