Fig. 4

Measurement of autophagy in HEK-293 cells treated with ZnO NPs. a Development of AVOs in HEK-293 cells. Detection of green and red fluorescence in AO-stained cells using flow cytometry. b Quantification of AVOs treated with ZnO NPs with AO. Cells were incubated with 0–25 μg/ml ZnO NPs for 24 h. *p < 0.05 versus control. The data are presented as the mean ± standard deviation of three independent experiments. c Immunofluorescence staining of the LC3 protein in HEK-293 cells treated with 20 μg/ml ZnO NPs for 24 h. d Western blotting for LC3-I, LC3-II, Beclin 1 and p62 in HEK-293 cells. Cells were treated with 0–25 μg/ml ZnO NPs for 24 h. e Ultrastructural changes observed in HEK-293 cells after ZnO NP treatment. Cells were treated with medium alone (untreated) or 20 μg/ml ZnO NPs for 24 h. The white arrowheads indicate the autophagic vacuoles and autolysosomes. N, nucleus