A., São Paulo, SP, Brazil) at concentrations of either 1 μM or 5 μM, according to the group. These concentrations were chosen based on a study by Scheper et al.17 who showed that ZOL can be found at these concentrations in the alveolar bone and saliva of patients under treatment with this drug. The culture medium

with the drug remained in contact with the cells in the incubator with 5% CO2 and 95% air at 37 °C for 24 h. Cell viability was evaluated using the methyltetrazolium (MTT) assay.18, 19 and 20 This method determines the activity of SDH enzyme, which is a measure of cellular (mitochondrial) respiration, BAY 80-6946 nmr and can be considered as the metabolic rate of cells. After 24 h of incubation of the cells in contact with DMEM alone (control group) or containing the two ZOL concentrations (experimental groups), the culture medium was aspirated and replaced by 900 μL of fresh DMEM plus 100 μL of MTT solution (5 mg/mL sterile PBS).

The cells were incubated at 37 °C for 4 h. Thereafter, the culture medium with the MTT solution was aspirated and replaced by 700 μL of acidified isopropanol solution (0.04 N HCl) in each well to dissolve the violet formazan crystals resulting from the cleavage buy Cyclopamine of the MTT salt ring by the SDH enzyme present in the mitochondria of viable cells, producing a homogenous bluish solution. After agitation and confirmation of the homogeneity of the solutions, three 100 μL aliquots of each well were transferred to a 96-well plate (Costar Corp.). Cell viability was evaluated by spectrophotometry as being proportional to the absorbance measured at 570 nm wavelength with an ELISA plate reader (Thermo Plate, Nanshan District, Shenzhen, Gandong, China). The values Flavopiridol (Alvocidib) obtained from the three aliquots were averaged to provide

a single value. The absorbance was expressed in numerical values, which were subjected to statistical analysis to determine the effect of ZOL on the mitochondrial activity of the cells. Total protein expression was evaluated as previously described.20 After 24 h of incubation of the cells in contact with DMEM alone (control group) or containing the two ZOL concentrations (experimental groups), the culture medium with ZOL was aspirated and the cells were washed three times with 1 mL PBS at 37 °C. An amount of 1 mL of 0.1% sodium lauryl sulphate (Sigma Aldrich Corp., St. Louis, MO, USA) were added to each well and maintained for 40 min at room temperature to produce cell lysis. The samples were homogenized and 1 mL from each well was transferred to properly labelled Falcon tubes (Corning Incorporated, Corning, NY, USA). One millilitre of distilled water was added to the blank tube. Next, 1 mL of Lowry reagent solution (Sigma Aldrich Corp.) was added to all tubes, which were agitated for 10 s in a tube agitator (Phoenix AP 56, Araraquara, SP, Brazil).