Fig. 6.

AOA-induced cell growth arrest and senescence of WI38 cells was not mediated by ROS signaling. (A) Effect of AOA on cellular ROS level. WI38 cells were treated with 3 mM AOA for 2 and 4 days (n=9). As a positive control, cells were exposed to 0.4 mM H₂O₂ for 2 h on the first 2 days. At the indicated time-period, cellular content of ROS was determined using DCFH-DA staining as detailed in the Materials and Methods. (B) Effect of ROS scavenger NAC on cell proliferation. Cells were pre-treated with vehicle or NAC for 1 h and then treated with or without 3 mM AOA for the indicated time-periods with medium changed at 2-day intervals. Cell numbers were assessed and calculated as described in Fig. 1A. (C) Effect of NAC on AOA-induced SA-β-gal activity. Cells were pre-treated with vehicle or 0.5 mM NAC for 1 h prior to treatment with AOA or H₂O₂ for the indicated time-period. As a positive control, WI38 cells were exposed to 0.4 mM H₂O₂ for 2 h on the first 2 days in the absence or presence of NAC. Senescent-like cells were evaluated by measuring SA β-gal activity as described in Fig. 3A. (D) Effect of chronic AOA treatment on cell morphology of WI38 cells. The cells were grown in the absence (control) or presence (+AOA, 6 day) of 3 mM AOA for 6 days, or in the presence of AOA for 6 days followed by removal of the AOA for an additional day (Removal of AOA). The H₂O₂-induced senescent WI38 cells (+H₂O₂, 6 day) serving as a reference became enlarged and flattened, which is a documented typical senescent morphology. All quantitative data are expressed as the mean±s.e.m. (n=3) of three independent experiments. Different lowercase letters indicate significant difference among treatment groups at the same time-point (P<0.05). Different uppercase letters indicate significant difference of the same treatment group at different time-points (P<0.05). Asterisk (*) designates a significant difference compared with the respective vehicle control at the same time-point (P<0.05).