The supernatants were collected for the assays. Activation of caspase-9 is based on hydrolysis of the substrate n-Acetyl-Leu-Glu-His-Asp7-amido-4-trifluoromethylcoumarin
(Ac-LEHD-AFC) by caspase-9, resulting in the release of fluorescent 7-amino-4-trifluromethyl coumarin (AFC) moiety, while hydrolysis of the peptide substrate acetyl-Asp-Glu-Val-Asp-7-amido-4-methylcoumarin (Ac-DEVD-AMC) by caspase-3, resulted in the release of the fluorescent 7-amino-4-methylcoumarin (AMC) moiety. Reaction were performed AZD0530 in vivo in buffer containing supernatant proteins (50 μg/sample for caspase-9 and 25 μg/Sample for caspase-3) and caspase substrates, at 37 °C for 2 h, followed by fluorimetric detection using the excitation and emission wavelengths of 400/505 nm and 360/460 nm for caspase-9 and caspase-3, respectively. The experimental data were evaluated using the analysis of variance (ANOVA), followed by the Dunnet test for the
comparison of the various treated groups with their controls, using the GraphPrism program, version 5.1 for Windows. The results were considered statistically significant at p < 0.05. The results showed that the congener BDE-99 inhibited cell proliferation after 24 and 48 h CT99021 supplier of incubation, showing significant effects at the higher concentrations tested (18.22 ± 6.42% and 41.77 ± 10.5% for 10 μM and 25 μM, respectively) after 24 h of exposure. A significant effect was observed for concentrations as low as 0.5 μM when the cells were exposed to the compound for 48 h (Fig. 1). Moreover, it was also demonstrated that the congener BDE-99 was able to induce a decrease in cell viability during both incubation periods for almost all the concentrations that lead to an inhibition of HepG2 cell proliferation (Fig. 2). These results demonstrated that there is a correlation between the effects observed in the first two experiments. Fig. 3 shows
FAD the effect of BDE-99 on the mitochondrial membrane potential (MMP). The MMP also changed after exposure to 10 and 25 μM of the compound for 24 h. This effect was intensified after 48 h of incubation, showing significant effects in concentrations as low as 0.5 μM. Similar results to those of the MMP assay were observed in the ROS accumulation test. Fig. 4 shows a significant increase in ROS accumulation after 24 h of incubation with BDE-99 at the highest concentration tested (25 μM). However when the effect was evaluated for 48 h, the exposure to 5 μM of the compound was sufficient to significantly increase ROS accumulation in the HepG2 cells. To better understand the mechanism by which BDE-99 induces cell death, we evaluated the exposure of phosphatidyl serine on the outer cell membrane by assessing the FITC-annexin-V positive cells. Fig.