Fig. 2

Effect of XWL-1-48 on ROS production and DNA damage, a, b After the cells treated with desired concentrations of XWL-1-48, GL331 and H₂O₂ for 24 h, ROS production was determined by flow cytometry. Columns represent the mean ± SD values obtained from three individual experiments; c MCF-7 cells were pre-treated with or without GSH (5 mM) for 1 h and then with XWL-1-48 (10 μM) for 24 h. The cell viability was determined by the MTT assay; d MCF-7 cells were treated with various concentration of XWL-1-48 or GL331 for 24 h, induction of γ-H2AX was determined by western blot; e MCF-7 cells were incubated with XWL-1-48 (10 μM) for indicated time point, γ-H2AX expression was measured; f Immunofluorescent staining of γ-H2AX in MCF-7 cells. Cells were incubated in 6-well plate overnight and then exposed to XWL-1-48 (1 μM) for 24 h. Cells were incubated with γ-H2AX antibody followed by incubation of secondary anti-rabbit IgG-FITC antibody (green). The nucleus was stained with PI (red). Images were captured using fluorescence microscope. Scale bar, 25 μm. g XWL-1-48 induced DNA damage by triggering ATM-related signaling pathways in MCF-7 cells. Expression levels of ATM, p-ATM, p-p53(ser15), p53 and p21 were analyzed. h, i MCF-7 cells were incubated with XWL-1-48 (10 μM) for the indicated time-point, the expression level of p-p53(ser15), p53 and p21 were determined by immunoblot. Data were shown as mean ± SD of three independent experiments. *p < 0.05, **p < 0.01 vs. control, respectively