Butein effect on AKT phosphorylation. (A) Cells were treated with 10 μg/ml of butein for 15 minutes. Relative phosphorylation levels of SRC, ERK and AKT in different breast cancer cell lines were presented in heatmap. (B) Representative data for phosphorylation levels of SRC, ERK, and AKT. Actin was detected as a loading control. (C) NMF analysis to categorize breast cancer cells based on butein-altered phosphorylation levels of SRC, ERK and AKT. (D-E) Cells were treated with 40 μg/ml of butein, 40 mM of NAC or 10 μM of LY294002 for 5 minutes. ROS levels were measured by incubating cells with H2DCF-DA for another 1 hour (D), and AKT phosphorylation was detected by anti-p-AKT antibody (E). (F) Cells were treated with NAC at 40 mM, LY294002 at 10 μM or butein at 10 μg/ml for 24 hours and then stained with Annexin V and PI. Apoptotic cells were measured by flow cytometry.