(A) ABR thresholds between Manf KO and wild type mice under CBA background are without significant differences throughout the frequency region (4–45 kHz), analysed at P56 (compare to age-matched Manf cKO B6 mice, Fig 1E). One-way ANOVA P-values: tone stimuli 4 kHz P = 0.458, 8 kHz P = 0.234, 16 kHz P = 0.407, 23 kHz P = 0.700, 32 kHz P = 0.881, 40 kHz P = 0.563, 45 kHz P = 0.632; click stimulus: P = 0.054. Animals per group: Manf KO CBA n = 6, wild-type CBA n = 3. Error bars show ± SE. (B, C) SEM images from the 45 kHz region of cochleas of WT and KO CBA mice show a comparable OHC hair bundle phenotype. (D) PTPRQ immunostaining coupled with phalloidin labeling shows the absence of PTPRQ expression in the 45-kHz OHC hair bundles in Manf KO CBA mice, consistent with the normal bundle morphology (compared to age-matched Manf cKO B6 mice, Fig 5A–D′). Abbreviations: ABR, auditory brainstem response; B6, C57BL/6J strain; CBA, CBA/Ca strain; cKO, conditional knock out; KO and Manf−/−, knock out; OHC, outer hair cell; PTPRQ, protein tyrosine phosphatase receptor type Q; SE, standard error of the mean; SEM, scanning electron microscopy; SPL, sound pressure level; WT, wild type. Scale bar: (B) 5 μm in all images.