Fig 2: Stereocilia morphology and MET function of cochlear hair cells are unaffected in Anxa4–/– mice. (A) Cochlear hair cell stereocilia of P5 wild type or Anxa4–/– mice were stained with anti-ANXA4 antibody (ABclonal) and imaged using confocal microscope. Stereocilia F-actin core was visualized by staining with TRITC-conjugated phalloidin. (B) Higher resolution confocal microscopy images show stereociliary tip localization of ANXA4 in P7 OHC and IHC. (C) Examination of hair bundle morphology of wild type or Anxa4–/– mice at P7 or P60 using SEM. (C’,C”) Higher resolution SEM images of OHCs and IHCs, respectively. (D) FM1-43FX uptake by OHCs and IHCs of P7 wild type or Anxa4–/– mice was examined using confocal microscope. Shown are images taken from the middle turn unless otherwise indicated. Scale bars, 10 µm in (A,D), 2 µm in (B,C’,C”), 5 µm in (C).

Fig 3: Vestibular function is unaffected in Anxa4–/– mice. The vestibular function of 2-month-old Anxa4–/– and wild type mice (n = 4) was evaluated by performing (A) head bobbing, (B) retropulsion, (C) circling stereotyped movement, (D) swimming, (E) tail hanging reflex, and (F) rotarod test. Five-month-old Cdh23 mutant mice (V2J) (n = 3) were included as positive control. ns, not significant; ***P < 0.001.

Fig 4: Cochlear hair cells and auditory function are unaffected in Anxa4–/– mice. (A) Cochlear hair cells of P7 Atoh1-GFP or Atoh1-GFP; Anxa4–/– mice were stained with anti-ANXA4 antibody (R&D Systems) and imaged using confocal microscope. Hair cells were labeled by nuclear GFP. (B) Cochlear hair cells of P60 wild type or Anxa4–/– mice were stained with anti-MYO7A antibody and imaged using confocal microscope. Shown are images taken from the middle turn. (C) Numbers of MYO7A-positive hair cells from different genotypes as indicated were calculated according to the results from (B). (D) Raw traces of ABR responses to click stimuli in 2-month-old wild type or Anxa4–/– mice. (E) The ABR thresholds for pure tone and click stimuli in 2-month-old wild type or Anxa4–/– mice. (F) The ABR thresholds for pure tone and click stimuli in 4–5-month-old wild type or Anxa4–/– mice. (G) Two-month-old wild type or Anxa4–/– mice were subjected to 2–20 kHz noise at 110 dB DSL for 2 hours, and ABR thresholds were measured pre-exposure and at different post-exposure time points as indicated. The numbers of animals used in each experiments are indicated in brackets. Scale bars, 20 µm in (A,B). ns, not significant.