Figure 7

Hmxbato increased reactive oxygen species (ROS) levels in lung tumor cells. Tumor (A549) and normal (BEAS-2B) lung cells were cultured for 4 h in the absence (negative control) or presence of hmxbato (3.8 µM and 19 µM) and subjected to the CM-H₂DCFDA incorporation assay. Bar graphs show the fluorescence intensity (ROS-positive cells) quantification of A549 (A) and BEAS-2B (B) cells at the different concentrations tested. Alternatively, the cells were incubated with hmxbato in the presence of NAC and were evaluated using a flow cytometer. (C) The histogram shows the release of ROS in control cells (gray line), cells treated only with 3.8 µM hmxbato (black line) and cells treated with 3.8 µM hmxbato in the presence of NAC (blue line). The measures obtained by flow cytometry were used to quantify the data shown in the graph. The graph shows the percentages of ROS release that were calculated considering the treatment with hmxbato in the absence of NAC as 100% ROS release. Data were expressed as mean ± standard deviation of experiments performed in triplicate. Three independent experiments were performed and over 10,000 cells were analyzed in each replicate. Statistical differences were determined using one-way ANOVA and Tukey's multiple comparison test. Significant differences with respect to control were considered when p < 0.1 (*), p < 0.01 (**), p < 0.001 (***) and p < 0.0001 (****).