Induction of ROS production and enzymes involved in ROS metabolism by CAP treatment in vitro. (A) Representative DCFH-DA staining (green) for ROS production in T24 cells (a–c) and 5637 cells (d–f) treated by CAP at 0, 150 and 300 µM for 48 h. Nuclei were stained by DAPI (blue). The scale bars for (a–f) are 20 μm; (B) Representative flow cytometry images for ROS detection by DCFH-DA in 5637 (a,c,e) and T24 cells (b,d,f) by CAP treatment at 0, 150 and 300 µM for 48 h; (C) Statistical analysis of cell number with positive DCFH-DA staining (a,b) and relative fluorescence of DCFH-DA using flow cytometry analysis (c,d), indicating significantly increased DCFH-DA positive BCa cells after CAP treatment. Results shown were mean ± SD of triplicate measurements and repeated three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001; (D) Western blot analysis revealed a strong upregulation of proteins involved in ROS metabolism: Catalase, FOXO3a, SOD2. GAPDH was used as a loading control. Cell types, CAP concentrations and protein masses were indicated.