Figure 4

S-phase arrest precedes onset of oxidative stress. Cell cycle distribution (A) and ROS levels (B) of U2OS cells were measured at different time points after RSV treatment. Cells were treated with 25 μM RSV for 0, 12, 24, 36, and 48 h, then the cell cycle distribution and ROS levels were measured by FACS analysis. (C) U2OS cells were pretreated with or without 10 mM NAC for 1 h, and then with or without 25 μM RSV for 24 h or 72 h. (D) Effect of GKT137831(GKT), a NADPH oxidase NOX1/NOX4 inhibitor, on ROS production. U2OS cells were incubated with RSV with or without GKT for 48 h and then ROS was measured by FACS analysis. *P < 0.05. (E) qRT-PCR analysis of various human NOX genes. U2OS cells were treated with RSV (50 μM) for different durations, the expression levels of NOX mRNAs were determined using RT-PCR.