Fig. 2

Exosomes protected hair cells against neomycin-induced damage in vitro. A–C Immunofluorescence staining was performed after neomycin damage (0.5 mM, 24 h) then with/without exosomes treatments (30 μg/ml, 24 h) in the middle turn of cochleae. A Immunofluorescence staining with Myo 7a (green), F-actin (red) and Hoechst (blue) after different treatments. Quantification of Myo 7a-positive hair cells per 100 μm in the middle turn of different groups. Scale bar, 20 µm. B Immunofluorescence staining with Myo 7a (green), Mito-SOX (red) and Hoechst (blue) after different treatments. The numbers and proportions of Mito-SOX and Myo 7a double-positive cells were quantified. Scale bar, 20 µm. C Immunofluorescence staining with Myo 7a (green), TUNEL (red), and Hoechst (blue) after different treatments. The numbers of TUNEL and Myo 7a double-positive cells were quantified. Scale bar, 20 µm. D–H HEI-OC1 cells were treated with neomycin (2 mM, 24 h), then with/ without exosomes (30 μg/ml, 24 h) treatment. D HEI-OC1 cells were labeled with Mito-SOX (red) and Hoechst (blue) and the relative fluorescence intensity was quantified. 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. Scale bar, 50 µm. G Cleaved CASP3 expression was detected by western blot in HEI-OC1 cells and was quantified by ImageJ software. H Analysis of apoptotic HEI-OC1 cells by flow cytometry. 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)