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. 2018 Jan 22;7:e31511. doi: 10.7554/eLife.31511

Figure 2. Immunolocalization of SLC7A8 in the mouse cochlea.

(A) Representative photomicrographs of cryosections of the base of the cochlea showing immunodetection for SLC7A8 (green) and s100 (red); and staining for DAPI (blue) or phalloidin (white) of wild type (upper row) and Slc7a8−/− mice (lower row). Scale bar, 100 µm. (B) On the left overlay image of a wild-type section indicating cochlea areas. Scale bar, 100 µm. On the right schematic drawing of the adult scala media adapted from Sanchez-Calderon et al. (2010). BC, border cells; CC, Claudius's cells; DC, Deiter's cells; HC, Hensen's cells; IC, intermediate cells; IHC, inner hair cells; IPC, inner phalangeal cells; Li, spiral limbus; MB, Basilar Membrane; OHC, outer hair cells; PC, pillar cells; RM, Reisner's membrane; SG, spiral ganglion; SL, spiral ligament; SV, stria vascularis; TM, tectorial membrane. (C) Quantification of SLC7A8 expression. Intensity of SLC7A8 immunofluorescence was normalized per mm2. Mean ±SEM from quadruplicates for each section, taken from apex, middle and basal cochlear turns of 4 wild-type (black), 3 Slc7a8+/− (green) and 4 Slc7a8−/− (blue) young (4- to 7-month-old) mice. Open and closed circles represent individual mice from C57BL6/J-129Sv or C57BL6/J backgrounds, respectevely. Unpaired Student’s t-test statistical analysis, p-values: *,≤0.05; **,≤0.01 and ***,≤0.001. (D) Quantification of SLC7A8 protein expression in the apex, middle and basal cochlear turns normalized per nuclei of young (2 month-old) (open bars) and old (12 month-old) (black bars) wild-type CBA mice. Data (mean ±SEM) were obtained from four cochlear sections obtained from three mice per group. Unpaired Student’s t-test statistical analysis, p-value: *,≤0.05.

Figure 2.

Figure 2—figure supplement 1. Quantification of transcripts in the Slc7a8−/− mouse cochlea.

Figure 2—figure supplement 1.

(A) Slc7a8 mRNA expression in cochlea at different ages in mice in MF1/129Sv genetic background. Expression levels, normalized by Hrpt1 gene expression, are represented as n-fold relative to control group (1 to 2-month-old). Values are presented as mean ±SEM of triplicates from pooled sample of 3 mice per group. Unpaired Student’s t-test statistical analysis (*: E18.5 versus other groups. ^: 1–2 months versus other groups) p-values: *,^ p≤0.05; **,^^ p≤0.01; ***,^^^p≤0.001. (B and C) mRNA levels were determined by RT-qPCR in the cochlea of 3 wild-type and 3 Slc7a8−/− young (3 to 7-month-old) C57BL6/J mice run in triplicates. Data (mean ±SEM) correspond to relative value normalized with Rplp0 gene expression. Unpaired Student’s t-test statistical analysis, p-values: *,≤0.05, **, &&≤0.01, ***, &&&≤0.001 and ****, &&&&≤0.0001. (B) Potassium voltage-gated channel subfamily Q member 5 (Kcnq5), (C) interleukin-1 (Il1) and interleukin-6 (Il6).
Figure 2—figure supplement 2. Progression of hearing phenotype of C57BL/6J Slc7a8 knockout mice.

Figure 2—figure supplement 2.

(A) Longitudinal analysis of Auditory Brainstem Response (ABR) thresholds in response to click stimuli in wild-type (Slc7a8+/+, white, n = 19), heterozygous (Slc7a8+/−, green, n = 13) and knockout (Slc7a8−/−, blue, n = 23) mice, through 2 to 5 months of age, and expressed as mean ±standard error. The significance of the differences was evaluated using ANOVA test, p<0.05 (Slc7a8−/− versus Slc7a8+/+ [*] or versus Slc7a8+/− [#]). (B) Pie chart showing the percentage of mice with normal hearing (all thresholds < 45 dB SPL, white), mild hearing loss (HL) (at least two tone burst threshold >45 dB SPL, orange) and severe HL (at least two tone burst threshold >60 dB SPL, red), within each genotype and age group.