Treatment with zinc oxide nanoparticles did not induce autophagy. A, Treatment with rapamycin induced autophagy as a positive control. H4-LC3-GFP cells were treated with rapamycin (0.2 μM) for 18 hrs. The nuclei were stained with Hoechst dye. The images were analyzed with a high-throughput microscope CellWoRx with a 10× objective. The average area of LC3-GFP quata are shown. The treatment of rapamycin led to an increase in autophagy. P < 0.001. Student T test. B, H4-LC3-GFP cells were treated with zinc oxide nanoparticles (Zn10 = 10 μg/ml, Zn30 = 30 μg/ml, Zn100 = 100 μg/ml) as indicated for 18 hrs. The nuclei were stained with Hoechst dye and the LC3-GFP dots were quantified using CellWoRx microscope with a 10× objective. The average intensities of LC3-GFP dots are shown. This experiment was repeated 2 times.