Fig. 5. Quantification of hair cell differentiation and rows in wild-type and COUP-TFI^-/- cochlea after DAPT treatment. (A) Real-time PCR analysis of myosin VIIa transcripts shows hair cell differentiation is dependent on DAPT dose. There is a significant difference in the COUP-TFI^-/- compared with wild type at the concentration of 5 µM (^**P<0.05). (B) Western blot confirmation of myosin VIIa protein expression after treatment with 5 µM DAPT shows an increase with DAPT treatment. The myosin band of Kaleidoscope prestained standard is used as control. (C) Hair cell row counts (myosin VIIa-positive inner and outer hair cells) along the length of the cochlear duct after culture with 5 µM DAPT. Both wild-type and COUP-TFI^-/- cochleae showed significant increases in hair cell rows after DAPT treatment (^*P<0.05), and COUP-TFI^-/- mutants had a significantly greater number than wild type in the apex (^**P<0.05). (D) Immunostained sections of DAPT-treated cochleae at the apical region show an increase in hair cell rows in the COUP-TFI^-/- mutants (c,d) compared with wild type (a,b; note, image in D part d is an extreme case of extra hair cells). Sections show hair cells expressing myosin VIIa (red) and nuclei counterstained with DAPI (blue). Scale bar: 20 µm.