Figure 4

Validation of temporal control and reversibility of Nkcc1 transcription. (a) The diagram shows that removal of DOX from mother’s diet suppresses Nkcc1 expression in P0-KD progeny during lactation. (b) qRT-PCR analysis revealed DOX regulation of Nkcc1 mRNA expression. At P35, cochlear Nkcc1 transcription is decreased in P0-KD mice vs. controls in comparison with other K⁺ circulation molecules as labeled. **Significant difference at p < 0.01 (Mann-Whitney test). NS: no significant difference (Mann-Whitney test) in P0-KD mice at P35. (c) In situ hybridization reveals that Nkcc1 mRNA expression was confined to the endolymphatic luminal side of the SV. (d) Magnified view of the image in (c). (e) The diagram reveals that, at 2 weeks post-administration of DOX in feed, Nkcc1 mRNA expression recovered fully and specifically in P35-reON mice at P49. (f) No significant difference in the expression of Nkcc1 in P35-reON and controls or vs. other K⁺ circulation molecules. NS: not significant (Mann-Whitney test) in P0-KD mice at P35. (g,h) Measure of ABR thresholds as readouts of hearing function. (g) Nkcc1 knockdown resulted in cochlear dysfunction in P0-KD mice with significant hearing loss (higher ABR thresholds) at three different frequencies, although it was milder than that observed in WL-KD mice. **Significant difference at p < 0.01 (Mann-Whitney test). (h) ABR thresholds at 24 kHz significantly decreased for P35-reON mice, which indicates hearing recovery at high frequency (coincident with the recovery of Nkcc1 transcription shown in panel f). *Significant difference at p < 0.05 (Dunn’s multiple comparison test).