In vitro studies
Animal care and use were in accordance with the ‘Guiding Directive for Humane treatment of Laboratory Animals'. The procedure was approved by the Chinese Science Academy Committee on Care and Use of Animals and performed using accepted veterinary standards. Post-natal day-2 transgenic mice containing the Math1-GFP
transgene were used in the in vitro
experiments. The mouse line was a generous gift from M. Charles Liberman, as previously described.39
Tissue culture and compound administration:
Dissection and culture of the mouse cochlear explant were performed as previously described.40
The tissues were cultured on poly-L-lysine-treated (Sigma-Aldrich, St. Louis, MO, USA) cover slides filled with 30μ
l of serum-free medium, which consists of DMEM/F12 basal medium supplemented with N2 and B27 solutions (Invitrogen, Carlsbad, CA, USA). The cover slides were placed in a 35-mm dish. To obtain a flat basilar membrane surface, the Reissner's membrane, spiral ligament, and stria vascular were carefully removed. After the tissues were attached to the slides, 1.5
ml of medium was added to the petri dish. Half of the medium was replenished every other day.
After the tissues were attached to the slides, they were incubated with the medium containing specific G9a/GLP inhibitors, 2μ
M BIX01294 or 2μ
M UNC0638 (Sigma-Aldrich), neomycin (1
mM) or a combination of both for designated times, as shown in Supplementary Tables S2
Quantification of the surviving hair cells:
To quantify the viable hair cells, the entire cochlea was divided into six segments of equal length, which were called segment 1, 2, 3, 4, 5, and 6 from apex to the base. The hair cells marked with Math1-GFP in the central field of view (360μ
m in each segment) were counted. Data are presented as means±S.E.M. ANOVA, the SNK-q test, or Student's t
-test were performed to analyse the compound effects. Differences between groups were considered statistically significant when P
Apoptotic cells were detected by the presence of nuclear condensation or fragmentation by using DAPI staining (Sigma-Aldrich), which was performed as previously described.41
This was also confirmed by TUNEL labelling (Fluorescein In situ
Cell death detection kit, Millipore, Bedford, MA, USA) according to the manufacturer's instruction. Quantification of the apoptotic cells in the cochlea was performed in the same way as described above.
GTTR and FM1-43FX uptake:
FM1-43 can enter hair cells as previously described.42, 43
To measure aminoglycoside uptake, the cochlear organs (neo group and pre-treatment group) were cultured for 24
h as described above. After rinsing with PBS, they were incubated with the same serum-free medium containing GTTR (10
mM) for 30
min or FM1-43FX (Molecular Probes, Eugene, OR, USA) for 3
min. After incubation, the cochlea was fixed for analysis using fluorescent microscopy.
Live-cell Imaging of mitochondrial membrane permeability:
The cochlear organs (neo group and pre-treatment group) were cultured and incubated in serum-free medium containing neomycin (1
mM) for 30
min, followed by serum-free medium containing TMRM (25
nM) for 30
min. The tissues were then washed with PBS before imaging analysis.
Samples were fixed in 4% paraformaldehyde in PBS for 2
h at room temperature, followed by rinsing three times in PBS. The tissues were then permeabilized in 0.2% Triton X-100 in PBS for 30
min at room temperature, blocked with 10% donkey serum in PBS for 1
h at 37
°C, and then incubated with the primary antibody (diluted in blocking solution) at 4
°C for 48
h. After washing away the primary antibody, the cultures were incubated with fluorescent dye-conjugated secondary antibody for 1
h at 37
°C to visualise the signal. The primary antibodies used were cleaved caspase-3 antibody (Cell Signaling Technology, Beverly, MA, USA, rabbit polyclonal, dilution 1
200) and anti-H3K9me2 antibody (Abcam, Cambridge, MA, USA, mouse monoclonal, dilution 1
200). The secondary antibodies were Alexa Fluor 555 donkey anti-mouse or rabbit IgG (Invitrogen, Carlsbad, CA, USA, dilution 1
400). The nuclei were labelled by DAPI staining. The images were captured using a Leica confocal microscope (Heidelberg, Germany).
Western blot analysis.
For western blot analysis, total protein was isolated from each of the 12 specimens using the AllPrep DNA/RNA/Protein Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. The protein concentrations were determined using a BCA protein kit (Beyotime, Jiangsu, China). In all, 50μ
g of the samples from each group were separated by 12% SDS-PAGE. After electrophorsis, the proteins were transferred to PVDF membranes (Millipore). The membranes were blocked with 5% non-fat dried milk in TBST for 1
h at room temperature. After washing, the primary antibodies, including cleaved caspaspe-3 antibody (dilution 1
1000), anti-H3K9me2 antibody (dilution 1
-actin antibody (Cell Signaling Technology, Beverly, MA, USA, rabbit polyclonal, dilution 1
1000), and anti-histone H3 antibody (Cell Signaling Technology, rabbit polyclonal, dilution 1
1000), were added into the blocking buffer overnight at 4
°C. After three times rinsing (10
min each) with TBST, the membranes were incubated with the blocking buffer containing the HRP-conjugated secondary antibody at a concentration of 1
5000 (Supersignal West, Pierce, Rockford, IL, USA) for 1
h at room temperature. The immunoreactive bands were visualised using an ECL kit (Pierce). To quantify the relative levels of the protein, images of specific protein bands were assessed by the grey-degree numerical value.
In vivo studies
Wild-type C57BL/6 mice were used for the in vivo
studies. All animal studies were in accordance with the ‘Guiding Directive for Humane treatment of Laboratory Animals. As shown in Supplementary Table S4
, we applied a retro-auricular surgical approach in 5-day-old mice following anaesthesia. To evaluate the otoprotection of G9a/GLP inhibition, the left ears were pre-treated with BIX01294 while the right ears were left untreated as the self-control. A piece of gelfoam (1
) soaked with BIX01294 (10
l) was positioned onto the round window niche through the small hole in the bulla, resulting in an approximate applied dose of 25μ
g of the drug in each pre-treated ear. The inferior skin incision was then closed with four silk sutures. Two days after administration of BIX01294, neomycin was injected subcutaneously once a day for 4 consecutive days. The neomycin was dissolved in saline at a concentration of 20
mg/ml so that a final dose of 200
mg of neomycin/kg of body weight was obtained by injecting 0.01
ml/g of body weight. Dosing of the neomycin was adjusted according to the accurate weight of the animals each day. Two weeks after exposure to neomycin, the hearing threshold was evaluated by ABR measurement.
The ABR analysis was performed in anaesthetised (100
mg/kg ketamine and 25
mg/kg xylazine sodium, i.p.) mice to measure the hearing threshold 2 weeks after the administration of neomycin. The hearing threshold was assessed at four frequencies (8, 16, 24, and 32
kHz) in a TDT system 3 (Tucker Davies Technologies, Gainesville, FL, USA). The traditional three electrodes (stimulus, reference, and grounding electrodes) were inserted subcutaneously at the vertex cranial, homolateral mastoid process, and nasal root, respectively. To prevent the influence from the other ear, the source microphone was positioned directly inside the external auditory canal during the sound acquisition. The threshold response was defined as the lowest response that could demonstrate a reproducible waveform. If there was ambiguity about the results, the ABR would be repeated the following day. The parameters of the evoked responses and acquired signal were as follows: duration of toneburst, 5
ms; rise–fall time, 0.5
ms; stimulus frequency, 21.37/s; stacking fold (superposition times), 500–1000; magnification, 20; bandpass, 0.3–3
kHz; sound intensity variation, 5
dB; amplitude of sound pressure level (SPL), 20–95
Scanning electron microscopy: The cochlea was perfused immediately with 4% paraformaldehyde after the mouse was anaesthetised. The tissues were then immersed in 2.5% glutaraldehyde for SEM. The SEM samples were post-fixed in 1% phosphate-buffered OsO4, dehydrated in a graded ethanol series, dried and mounted on to an aluminium sheet, and sprayed with gold–palladium. SEM was performed using a Philips XL-30 (Eindhoven, the Netherlands) SEM apparatus.
Immunohistochemistry: Immunohistochemistry was performed similarly as described above. Hair cells were marked with polyclonal anti-myosin 7a (Proteus Biosciences, Ramona, CA, USA). The apoptotic bodies were detected by Hoechst 33342 (Sigma-Aldrich) to identify the condensed or fragmented nuclei.
Statistics: The results were presented as means±S.E.M. Student's t-test was performed to determine statistical significance. The results were considered significant when P<0.05 between the groups.