Figure 5. PS induces oxidative stress in vitro and in vivo.

NSCLC cells were treated with PS at the indicated concentrations for 1h. A, Left: PS induced ROS in A549 and H358 cells, but not in H23 cells as determined by DCFDA staining. Right: The effect of N-acetylcysteine (NAC, 10mM for 4h) pretreatment on apoptosis induction by PS. B, PS induced mitochondrial ROS stress in all three NSCLC cell lines as determined by MitoSOX Red staining. C, PS caused the collapse of mitochondrial membrane potential (Δψm), as indicated by the increased JC-1 fluorescence relative to the control. D, Mitochondria-depleted A549 cells (ρ⁰) showed resistance to PS. Left, upper: Immunoblotting of control and ρ⁰ A549 cell lysates for the specific marker mitochondrial protein cytochrome c oxidase subunit IV (COXIV). Left, lower: ρ⁰ and control A549 cells were treated with PS for 24h and cell viability was determined by trypan blue exclusion. Depletion of mitochondria substantially increased the resistance of A549 cells to the cytotoxic effect of PS. Right: ρ⁰ A549 cells were much more resistant to PS-induced apoptotic cell death, as measured by Annexin V/PI staining and flow cytometry.