Fig. 6. Overexpression of YAP initiated supernumerary HCs.

a After neomycin exposure for 6 h, the cochlear explants were cultured with exogenous Ad-GFP/Ad-Yap-GFP for 48 h, and the transfection efficiency based on the GFP fluorescence was analyzed in Sox2+ SCs in the SR. b, c GFP+/Sox2+ cells were counted, and the results showed that the transfection efficiency increased with the increasing adenovirus concentrations (10⁷, 10⁸, 10⁹, 3 × 10⁹, and 10¹⁰ PFU/ml). d The numbers of Sox2+ SCs treated with different concentrations of adenovirus were calculated. It was found that the number of SCs decreased slightly at high concentration. e, f To upregulate the expression of Yap, the cochlear explants after neomycin insult for 6 h were treated with Ad-Yap-GFP for 48 h. One-sample t test was performed and showed that YAP was significantly increased with a concentration of 3 × 10⁹ PFU/ml adenovirus, and the distribution of YAP in the nucleus was significantly increased. g The relative mRNA expression level of Hippo signaling and downstream target genes after Ad-Yap-GFP treatment, and two-way ANOVA followed by Sidak’s multiple comparison test was performed to show the significant differences. h–k After neomycin exposure for 6 h, the cochlear explants were cultured with Ad-mus-Yap1 or Ad-null for 7 days, and 10 μM EdU was added the whole time to trace proliferating cells. In the control group with Ad-null treatment, no proliferating HCs or SCs were detected. In the Ad-mus-Yap1 treatment group, there were significantly more EdU+ SCs and HCs from the apical to basal turn analyzed with two-way ANOVA followed by Sidak’s multiple comparison test. Compared with controls, the number of HCs was significantly increased from the apical to the basal turn analyzed with two-way ANOVA followed by Sidak’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Data are shown as the mean ± SEM. Scale bars = 20 μm in (b) and (h), Scale bars = 10 μm in (e).