Fig. 5

Autophagy was necessary for exosome-mediated otoprotection. A–C Cochlear explants were treated with 3-MA (5 mM) for 16 h. Cell survival, oxidative stress and apoptosis of hair cells were respectively detected by phalloidin staining (A), Mito-SOX Red (B), and TUNEL assay (C) following co-culture with exosomes after neomycin exposure. The number of F-actin and Myo 7a double-positive cells, Mito-SOX and Myo 7a double-positive cells and TUNEL and Myo 7a double-positive cells per 100 μm in the middle turn of different groups were quantified. Scale bar, 20 µm. D–H HEI-OC1 cells were treated with 3-MA (5 mM) for 16 h to inhibit autophagy. D HEI-OC1 cells were labeled with Mito-SOX (red), and the relative fluorescence intensity was quantified after different treatments. Scale bar, 20 µm. E TUNEL and Hoechst double staining and F Cleaved CASP3 and Hoechst double staining were performed to detect the percentage of apoptotic HEI-OC1 cells after different treatments. Scale bar, 50 µm. G Cleaved CASP3 expression was detected by western blot in HEI-OC1 cells treated with exosomes and/or 3-MA following neomycin insults and was quantified by ImageJ software. H Analysis of apoptotic HEI-OC1 cells by flow cytometry after different treatments. The results were representative of the data generated in at least three independent experiments. The data were presented as mean ± s.d. n.s., not significant; *P < 0.05; **P < 0.01 by one-way ANOVA (A–H)