Fig. 5: TMC1 localization in OHC stereocilia is affected by the Loxhd1^Delta allele.

a TMC1 localization by immunofluorescence (anti-HA) in P7 OHC hair bundles (Phalloidin) along the tonotopical axis in Tmc1^HA/HA with Loxhd1^+/+, Loxhd1^Delta/+, or Loxhd1^Delta/Delta. TMC1^+/+; Loxhd1^Delta/+ was used as “non-HA” negative control. Scale bars = 4 µm. b Whole hair bundle TMC1-HA fluorescence intensity quantification on P7 apical, medial, and basal OHCs of Tmc1^HA/HA with Loxhd1^+/+, Loxhd1^Delta/+, or Loxhd1^Delta/Delta animals. Then intensity values were normalized to the mean fluorescence intensity of apical Tmc1^HA/HA; Loxhd1^+/+ OHCs. Per group: n_cells ≥ 27. The experiment was replicated twice. Two-way ANOVA followed the Tukey test. c SUB-immunogold-SEM anti-HA detects TMC1-associated gold beads (10 nm, arrowheads) at OHC row 2 and 3 tips of Loxhd1^+/+ and Loxhd1^Delta/Delta P7 medial OHCs. The experiment was performed once. Scale bars = 200 nm. d Distance between TMC1-gold beads to the stereocilia tips in row 2 and 3 stereocilia measured from SUB-immunogold-SEM micrographs in Loxhd1^+/+ and Loxhd1^Delta/Delta P7 OHCs. Quantified OHCs were from pooled tonotopic positions distributed as follow: 35% apical, 27% middle, and 38% basal. Per group: P7 row 2: n_cells ≥ 38; n_stereocilia ≥ 113; row 3: n_cells ≥ 39; n_stereocilia ≥ 75; The experiment was performed once. e, f Stereocilia classification based on the TMC1-gold distribution pattern: with at least one gold bead present regardless of its position, with at least one gold bead in the first 100 nm, with at least one gold bead below 100 nm, and without any gold beads. Row 2 (e): per group: n_cells ≥ 33; row 3 (f): per group: n_cells ≥ 35; Represented as mean % ± SEM. Two-way ANOVA followed by Tukey’s multiple comparisons tests. For all experiments. ns (non significative) = p > 0.05; *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The exact p values are presented in Supplementary Table 1. Source data are provided as a Source Data file.