Figure 6.

The role of zinc ions in ZnO NPs-induced osteosarcoma cell death. U-2OS cells were pre-incubated with EDTA or CaCl₂ for 1 h, and then treated with ZnO NPs (50 μg/ml) for 24 h. (a) The concentration of intracellular zinc ions detected by ICP-MS for U-2OS and Saos-2. (b, f) Cell viability analyzed by CCK-8 in the presence and absence of EDTA (b) or CaCl₂ (f) with varying concentrations. (c, d) Cell viability analyzed by CCK-8 (c) and LC3 and pro caspase-3 levels detected by western blotting (d) after the cells were exposed to varying concentrations of ZnCl₂. (e) The concentration of intracellular zinc ions after the osteosarcoma cells were under the equivalent inhibitory effect of ZnO NPs and ZnCl₂. (g) The concentration of zinc ions in the supernatant released from ZnO NPs with or without CaCl₂ detected by ICP-MS. (h) The concentration of intracellular zinc ions detected by ICP-MS in the presence and absence of EDTA or CaCl₂. (i) LC3 and pro caspase-3 levels was detected by western blotting in the presence and absence of EDTA or CaCl₂. 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.