ROS mediates AA005-induced cell death of SW620 cells. (A) SW620 cells were treated with or without 1 μM AA005 for indicated times. Complex I subunits NDUFS1 and NDUFA10 were detected by western blot, standardized to actin. (B−C) SW620 cells were treated with or without AA005 at 1 μM for 1 h, 4 h or 12 h. DCFDA staining was then analyzed on flow cytometry (B); quantified data: (C). Experiments were repeated 3 times with the same results. Results show mean ± S.D. *P < 0.05, **P < 0.01, compared with vehicle-only control. (D, E) After pretreatment with or without 1 mM NAC for 2 h, cells were treated with or without AA005 at 1 μM for 60 h. Annexin-V/PI double-stained cells were measured on flow cytometry. Panels show percentages of Annexin-V+ or PI+ cells (D). Dead cells (E). Each column shows mean ± S.D. of triplicates in an independent experiment. **P < 0.01, vs AA005-treated group.