Treatment with titanium dioxide nanoparticles led to increases in the autophagosome flux. H4-LC3-GFP cells were treated with titanium dioxide nanoparticles (Ti10 = 10 μg/ml, Ti30 = 30 μg/ml, Ti100 = 100 μg/ml) both in the absence and presence of N-acetylcysteine (2.5 mM) or E64d (5 μg/ml) as indicated for 18 hrs. The nuclei were stained with Hoechst dye and the LC3-GFP dots were recorded using a high-throughput microscope CellWoRx with a 10× objective. The average intensities of LC3-GFP dots are shown. The images were quantified using VHSscan and VHSview image analysis software (Cellomics). The differences between control and Ti10, Ti30 or Ti100 and that of E64d and E64d + Ti1100, are highly significant with p < 0.0001 (***, Student T-test). This experiment was repeated 2 times.