Experimental Animals
All animal experiments were approved by the Institutional Animal Care and Use Committee of the First Affiliated Hospital of Wenzhou Medical University . Care and use of animals followed the guidelines in the NIH Guide for the Care and Use of Laboratory Animals. All efforts were made to minimize pain. After comprehensive consideration of various factors such as noise sensitivity, auditory threshold and aging hearing loss, Wistar rats were used as experimental animals in this experiment. Wistar rats (female; 3 months; n=10) were provided by the animal facility of the Shanghai SLAC Laboratory Animal Co.,Ltd (Shanghai, China), and were housed in standard cages and maintained on a 12/12 h light/dark cycle at a controlled temperature (20-22 °C) and humidity (50 ± 5%). Rats had free access to standard rodent chow and filtered water at all times. After adapting to the environment for one week, Wistar rats were randomly divided into 2 groups (n=5 of each group): noise exposed group (NIHL group) and non-exposed group, and the auditory brainstem response before and after noise exposure was recorded.
Noise exposure
Rats were exposed to low-frequency, mid-frequency, and high-frequency noise for one day in a custom-made sound exposure room (Acoustical Laboratory, Naval Medical Research Institute, Shanghai, China) equipped with an internal light source and ventilation system, and compare with the unexposed group. Animals were conscious, unrestrained and had free access to food and water throughout the noise exposure period.
Auditory Brainstem Responses (ABR)
ABR analysis for the auditory evaluation was performed in rats used for NIHL study[24]. Wistar Rats were anesthetized with ketamine (50 mg/kg) and xylazine (6 mg/kg) cocktail by intraperitoneal injection and placed on a 37°C thermostatic heating pad. Electrodes were placed on the vertex (non-inverting) and on ipsilateral mastoid (inverting) and hind limb (ground). ABRs were collected through Intelligent Hearing Systems (IHS, Miami Florida, USA). ABRs were filtered, amplified and digitized (1024 presentations, 40 kHz sampling rate, 30-3000 Hz, 100X) in response to tone bursts presented at 6, 12, 16, 20, 24 and 32 kHz (1 ms rise/fall, cosine gated, 5 ms duration, 21/s).
Cell culture
The cochlear auditory cell line HEI-OC1, purchased from Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Science (CAS; Shanghai, China), were treated with DMEM medium supplemented with 10% FBS. All cells were kept in a 37 °C incubator with 5 % CO2.
Construct of lentivirus and cell transfection
FBXO3 shRNA (shFBXO3) were designed and synthesized from Shanghai Genechem Co., LTD (Shanghai, China). The corresponding sequences were cloned into the lentiviral vector to construct recombinant lentiviral Lv-shFBXO3. Lv-shFBXO3 was used to inhibit FBXO3 expression in HEI-OC1 cells. FBXO3 expression level in selected clones was verified by western blot analysis.
Western bolt analysis
HEI-OC1 cells were lysed with RIPA Lysis and Extraction Buffer (Thermo Fisher Scientific). The protein concentration was determined using a BCA Protein Assay Kit (Abcam, Cambridge, MA, USA). Then, 40 μg protein was separated on 10% SDS‐PAGE and transferred onto PVDF membranes (Merck, Darmstadt, Germany). The membranes were blocked with 5% skimmed milk and probed with primary antibodies [FBXO3 (ab224603, 1:1000, Abcam), ATG10 (ab240901, 1:1000, Abcam), LC3 A/B (ab128025, 1:1000, Abcam) and β‐actin (ab179467, 1:5000, Abcam) at 4°C overnight. Next, secondary antibody Goat Anti-Rabbit IgG H&L (HRP) (ab205718, 1:5000, Abcam) was added. The obtained bands were detected using the ECL system, and protein bands were analysed using Quantity One software from Bio‐Rad.
Tissue preparation and immunohistochemistry assay
The cochleae from NIHL group and non-exposed group rats were fixed with 4% paraformaldehyde (PFA) overnight. Samples were decalcified in 10% EDTA for 5 to 7 days and then dehydrated in 30% sucrose solution for 12 h, sectioned into 10-μm-thick slices across the modiolus, and stored at -80 °C until immunohistochemistry staining.
For the immunohistochemistry assay, the sections were treated with 3% H2O2 and then incubated in a blocking buffer with 5% BSA and 0.3% Triton X-100 (Sangon Biotech, Shanghai, China) for 60 min at room temperature (RT). Anti-LC3B antibody (ab128025, 10 µg/ml, Abcam) at 4°C overnight, washed 3 times with PBS for 10 min, and then incubated with secondary antibody (ab205718, 1:5000, Abcam) at RT for 40 min. DAB chromogenic reagent was added and incubated for 10 min. The nuclei were further stained with hematoxylin.
Immunofluorescence
HEI-OC1 cells were fixed with 4% PFA for 20 min, and then washed with PBS. The cells were incubated with anti-FBXO3 antibody (1:200; ab224603, Abcam) overnight at 4 ℃, and then incubated with the second antibody (1:5000) for 1 h after PBS washing 3 times. The nuclei were stained with DAPI (Sigma-Aldrich) for 20 min. A fluorescence microscope was used to observe and record the images.
Co-IP assay
Co-IP was carried out with a Pierce™ Co-Immunoprecipitation kit (Thermo Fisher Scientific) as per the manufacturer’s protocol[25]. In brief, HEI-OC1 cells were lysed on ice with lysis buffer for half an hour, centrifuged for 12 min and the supernatant was discarded. 400 μl supernatant with 1mg protein was incubated with 10 μg anti-FBXO3, anti-ATG10 or anti-IgG antibodies for 12 h at 4°C. Beads were washed, eluted in sample buffer, and boiled for 10 min at 100°C. Finally, immune complexes were stained with Coomassie Blue and analyzed using SDS-PAGE.
Statistical analysis
Data were represented as mean ± SD. The difference between two groups was compared using two-tailed student’s t-test, or one-way analysis of variance (ANOVA) followed by the Scheffé test. A value of p < 0.05 was considered statistically significant. Data statistical analyses was carried out by the SPSS software version 16.