Then, the cells were treated for 12- and/or 24-h at concentrations of 2.5, 5 and/or 10 μg/ml, corresponding to: 6.1, 12.2 and 24.4 μM for AC-4; 5.3, 10.6 and 21.2 μM for AC-7; 5.8, 11.6 and 23.2 μM for AC-10; 6.0, 12.1 and 24.1 μM for AC-23, respectively. Fluorouracil clinical trial The trypan blue exclusion test was performed before each experiment described below to assess cell viability. The negative control was treated with the vehicle (0.1% DMSO) used for diluting the tested substances. Amsacrine (m-AMSA, 0.3 μg/ml [0.8 μM], Sigma Chemical Co. St Louis, MO, USA) or doxorubicin (0.3 μg/ml [0.6 μM], Sigma Chemical Co. St Louis, MO, USA) was used as the positive control. The concentrations of ATZD

used here were based on their IC50 value in this cell line (3.1 μg/ml for AC-4, 5.3 μg/ml for AC-7, 3.6 μg/ml for AC-10 and 2.3 μg/ml for AC-23) as buy MG-132 previously described ( Barros et al., 2012). Cell proliferation was determined using the Trypan blue dye exclusion test.

After each incubation period, the cell proliferation was assessed. Cells that excluded trypan blue were counted using a Neubauer chamber. Twenty microliters of 5-bromo-20-deoxyuridine (BrdU, 10 mM) was added to each well and incubated for 3 h at 37 °C before 24-h of drug exposure. To assess the amount of BrdU incorporated into DNA, cells were harvested, transferred to cytospin slides (Shandon Southern Products Ltd., Sewickley Pennsylvania, USA) and allowed to dry for 2 h at room temperature. Cells Rebamipide that had incorporated BrdU were labelled by direct peroxidase immunocytochemistry using the chromogen diaminobenzidine. The slides were counterstained with hematoxylin, mounted and put under a cover slip. A light microscopy (Olympus, Tokyo, Japan) was used to determine BrdU-positivity. Two hundred cells per sample were counted to determine the percent of BrdU-positive cells. Untreated or

ATZD-treated HCT-8 cells were examined for morphological changes under a light microscopy (Metrimpex Hungary/PZO-Labimex Model Studar lab). To evaluate any alterations in morphology, cells from the cultures were harvested, transferred to a cytospin slide, fixed with methanol for 30 s, and stained with hematoxylin–eosin. Cells were pelleted and resuspended in 25 μl of PBS. Then, 1 μl of aqueous acridine orange/ethidium bromide solution (AO/EB, 100 μg/ml) was added and the cells were observed under a fluorescence microscope (Olympus, Tokyo, Japan). Three hundred cells were counted per sample and classified as viable, apoptotic or necrotic (McGahon et al., 1995). The integrity of the cell membrane was evaluated using the exclusion of propidium iodide (2 μg/ml, Sigma Chemical Co. St Louis, MO, USA). Cell fluorescence was determined by flow cytometry in a Guava EasyCyte Mini System cytometer using CytoSoft 4.1 software (Guava Technologies, Hayward, California, USA). Five thousand events were evaluated per experiment and the cellular debris was omitted from the analysis.