Fig. 4

Abnormal accumulation of autophagosomes in ZnO NPs-treated PC12 cells. Effects of well-known cell death inhibitors on the cytotoxicity of ZnO NPs by PI staining (a) and by CCK8 assay (b). PC12 cells were treated with 15 μg/mL ZnO NPs in the presence or absence of various cell death inhibitors, Z-VAD (Z-VAD-fmk, caspase inhibitor, 20 μM), Necrostatin 1 (Nec-1, RIPK1 inhibitor, 20 μM), Necrosulfonamide (NSA, MLKL inhibitor, 1 μM), 3-Methyladenine (3-MA, autophagosome formation inhibitor, 2 mM), Chloroquine (CQ, inhibitor of mature autophagosomes fusion with lysosomes, 10 μM), Ferrostatin-1 (Fer-1, lipid peroxidation inhibitor, 1 μM), Liproxstatin-1 (Lip-1, another lipid peroxidation inhibitor, 500 nM). c Representative TEM images of autophagosomes. PC12 cells were treated with 15 μg/mL ZnO NPs. Red arrows indicated autophagosomes. d Flow cytometry analysis of autophagosomes by MDC staining in ZnO NPs-induced PC12 cells for 6 h with or without 3-MA and CQ pre-treatment for 1 h. Data from three independent experiments were expressed as the means ± SD. ***p < 0.001 compared with the untreated control. e Effects of ZnO NPs on the expression of autophagy-related proteins. LC3B, Beclin 1 and p62 expression were examined by Western blotting. f Western blotting analysis of LC3B and p62 expression levels in PC12 cells treated with 15 μg/mL ZnO NPs for 6 h in the presence or absence of CQ pre-treatment for 1 h. Images were representative of at least three independent experiments, and the densitometry of those bolts was shown in Figure S8A and B