Fig. 3

Exosomes improved autophagy in cochlear hair cells and HEI-CO1 cells. A Expression of LC3, SQSTM1/p62 and BECN1 in cochlear explants treated with neomycin (0.5 mM, 24 h) and/or exosomes (30 μg/ml, 24 h) was evaluated by western blot and quantified by ImageJ software. B TEM analysis was used to evaluate autophagy in cochlear hair cells, and the numbers of autophagic vacuoles were quantified. Scale bar, 1 µm (up), 200 nm (down). C Immunofluorescence staining with Myo 7a (blue) in cochleae from CAG-RFP-EGFP-LC3 mice. The numbers of autophagosomes (yellow) and autolysosomes (red-only puncta) per cell were quantified. Scale bar, 10 µm. D Expression of LC3, SQSTM1/p62 and BECN1 in HEI-OC1 cells treated with neomycin and/or exosomes was evaluated by western blot and quantified by ImageJ software. E Autophagy in HEI-OC1 cells was detected by TEM, and the numbers of autophagic vacuoles were quantified. Scale bar, 2 µm (up), 500 nm (down). F HEI-OC1 cells were infected with mRFP-GFP-LC3 (tfLC3) and then treated with neomycin and/or exosomes. The numbers of autophagosomes (yellow) and autolysosomes (red-only puncta) per cell were quantified. Scale bar, 10 µm. G Autophagy flux was evaluated by western blotting for LC3 with or without CQ (20 μM). Autophagy flux assay was used in HEI-OC1 cells treated with exosomes at different time points, and LC3-II (CQ-Ctrl) was quantified by ImageJ software. The results were representative of the data generated in at least three independent experiments and presented as mean ± s.d. n.s., not significant; *P < 0.05; **P < 0.01 by one-way ANOVA (A-G)