Butein effects on ROS generation. (A) Butein alteration of ROS level. 100% stacked bars indicate ROS levels in the cells treated with butein or not. Cells were treated with butein at 10 μg/ml for 5 minutes and incubated with H2DCF-DA for another 1 hour. ROS levels were measured using flow cytometry. Experiments were independently repeated three times. Red arrows indicate ROS levels in HER2+ breast cancer cells. (B) Representative data for butein-altered ROS levels in HER2+ breast cancer cells either sensitive (BT-474) or resistant (HCC-1419) to butein. Negative control (Neg.) indicates no treatment of ROS indicator. Left triangles indicate the increase of ROS levels. (C) Correlations between percentages of reduced ROS levels and percentages of apoptotic cell death. A red circle indicates butein-resistant and luminal HER2+ breast cancer cells. (D-E) Butein effect on cell cycle profiles. Cells were treated with 10 μg/ml of butein for 24 hours and then stained with PI. Data present the mean ± SD for altered percentages of subG1 populations in different breast cancer cells treated with 10 μg/ml of butein for 24 hours (D). Cell populations at different stages of cell cycle were presented as 100% rate (E). Experiments were performed in triplicate.