Fig. 6

Exosomes promoted endocytosis, which is required for exosomes to upregulate autophagy of hair cells. A PKH26-labeled exosomes co-localized with endosomes, which were immunofluorescence stained with EEA1 in HEI-OC1. Scale bar: 20 µm. B RT-qPCR was performed to detect the endocytosis-associated mRNA levels of HEI-OC1 cells after treatment with exosomes (30 μg/ml, 24 h). C The protein expression of CAV2, EEA1 and LC3 was detected by western blot and was quantified by ImageJ software. D The expression of EEA1 was detected by immunofluorescence staining, and the fluorescence intensity was quantified by ImageJ software. Scale bar, 50 μm. E Expression of LC3, SQSTM1/p62 and BECN1 were detected by western blot after exosome treatment with or without pre-treatment of dynasore (80 μM, 4 h) or cytochalasin D (2 μM, 30 min) and were quantified by ImageJ software. F Autophagy flux assay of HEI-OC1 cells treated with exosomes without endocytosis inhibition, and LC3-II (CQ-Ctrl) was quantified by ImageJ software. G, H Autophagy flux assay of HEI-OC1 cells treated with exosomes with endocytosis inhibition by either dynasore (G) or cytochalasin D (H), and LC3-II (CQ-Ctrl) was quantified by ImageJ software. I HEI-OC1 cells were infected with mRFP-GFP-LC3 (tfLC3) and then treated with exosomes with/without dynasore or cytochalasin D. The numbers of autophagosomes (yellow) and autolysosomes (red-only puncta) per cell were quantified. Scale bar, 10 µm. 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 Student’s t-test (B–D) or by one-way ANOVA (E–I)