Figure 8.

The role of ROS in ZnO-induced osteosarcoma cell death. U-2OS cells were pre-incubated with NAC (10 nM) for 1 h, 3-MA (10 nM) for 2 h and then treated with ZnO NPs (50 μg/ml) for 24 h. (a) Cells were loaded with DCFH-DA and then the levels of ROS were analyzed by flow cytometry. (b, c, d, e) Cell viability detected by CCK-8 in the presence and absence of NAC with varying concentrations (b), apoptosis (c) and cell cycle (d) analyzed by flow cytometry in the presence and absence of NAC, and cell cycle related proteins detected by western blotting (e). (f) The levels of AO staining in the presence and absence of NAC analyzed by flow cytometry. (g) LC3 levels detected by western blotting and quantified by densitometric analysis relative to β-actin. (h) The levels of ROS analyzed by flow cytometry after the cells were labeled with DCFH-DA for 30 min. Statistically significant differences were evaluated using the paired Dunnett`s t-test or one-way ANOVA. Results were expressed as the mean ± S.D. from three independent experiments. *P<0.05 versus control, #P<0.05 versus ZnO NPs treatment.