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. 2019 Jul 28;9(19):5577–5594. doi: 10.7150/thno.34663

Figure 3.

Figure 3

ROS induced by DJ-1 downregulation are responsible for anti-CRC effects of CPX. A. ROS level was analyzed by DCFH-DA staining via a fluorescence microplate Reader (Thermo scientific) in CRC cells treated with the indicated concentrations of CPX. Statistical significance compared with respective control groups. B. MitoSOX Red was used to evaluate ROS level in mitochondria upon CPX treatment. Scale bar, 25μm. C. ROS level was determined by flow cytometry in CRC cells treated with or without 20 μM CPX for 24 hours overexpressing Myc-DJ-1 and Vector. D. Colony formation assay of CRC cells treated with the indicated concentrations of CPX in the presence or absence of 5mM NAC. E. EdU assay of CRC cells treated with or without 5mM NAC in the presence or absence of 20 μM CPX for 24 hours. The EdU incorporation was quantitated. Scale bar, 100 μm. F. The CCK8 assay determined cell viability of CRC cells treated with the indicated concentrations of CPX in the presence or absence of 5mM NAC for 24 hours. G. Immunoblot analysis of PARP expression in HCT116 and SW480 cells treated with CPX (20 μM) or vehicle and/or with NAC (5mM). H. HCT116 and SW480 cells treated with CPX (20 μM) or vehicle and/or with NAC (5mM) were fixed, stained with Annexin V/PI, and then analyzed by flow cytometry. The development of apoptosis was quantitated. Data are means ± s.d. and are representative of 3 independent experiments. *, P < 0.05, **, P < 0.01, ***, P < 0.001.