Noise-induced hearing loss in G6PD transgenic mouse

[Description of methods used for collection/generation of data] ABR DATA: ABR recordings were performed on a TDT RZ6 evoked potential workstation (Tucker‐Davis Technologies, Alachua, FL, USA), as reported by Cediel et al. (2006). ABR test were performed before and after exposure to noise in G6PD transgenic and wild type mice. In brief, mice were anesthetized with ketamine (100 mg/kg; Imalgene 1000; Merial, Lyon, France) and xylazine (10 mg/kg; Rompun 2%; Bayer, Leverkusen, Germany) by intraperitoneal injection and the ABR tests were performed in a sound‐attenuating chamber. Two different sound stimuli, clicks and tone bursts, were generated with SigGenRP software (Tucker‐Davis Technologies). Stimuli were calibrated using SigCal software and an ACO Pacific 1⁄4‐inch microphone. Click stimuli were 0.1 ms and toneburst (4, 8, 16, 28, and 40 kHz) stimuli were 5‐ms duration (2.5 ms each for rise and decay, without plateau). The response was analyzed with BioSigRP software (Tucker‐Davis Technologies). Stainless steel needle electrodes were placed at the vertex and ventrolateral to the left and right ears for recording and a tweeter in open field configuration to deliver acoustic stimuli. Hearing thresholds were established at the lowest SPL level that produced a noticeable ABR five peaks wave and evoked a peak‐to‐peak voltage 2 SD above the mean background activity. Wave amplitudes, latencies, and inter‐wave latencies were determined at 70 dB SPL click stimulation. qPCR DATA: obtained by Real-Time PCR and analyzed by QuantStudio™ Real-Time PCR software 1.3. Cochleae were isolated from vestibules and frozen in RNAlater ® solution. Cochlear RNA extraction from pooled cochlea (n = 3 mice per experimental group), quality determination, and cDNA generation were performed as reported by Celaya et al. (2019). Quantitative amplification was performed in triplicate on an Applied Biosystems 7900HT Real‐Time PCR System using either commercial TaqMan probes or gene‐specific primers (Tables ​(Tables11 and ​and2).2). Data were collected after each amplification step and analyzed with SDS 2.2.2 software (Applied Biosystems, Foster City, CA, USA). The 18s gene was used as a housekeeping gene and the n‐fold differences were calculated using the 2–ΔΔCt method. Total G6PD expression levels are represented as the sum of the 2–ΔCt for the murine and human primers. WB data: Whole cochleae protein extracts were prepared from 3 mice as described (Sanchez‐Calderon et al., 2010). An equal volume of extracts was resolved using SDS‐PAGE, followed by transfer to PVDF membranes (0.2 μm; Bio‐Rad Laboratories, Hercules, CA, USA) using the Bio‐Rad Trans Blot TURBO apparatus. Membranes were blocked with 5% BSA or non‐fat dried milk in 0.075% Tween, 1 mM TBS, and incubated overnight with the following antibodies: HO-1 1:1000 Conejo Pc Millipore/ #AB1284 SOD2 1:1000 Conejo Pc Millipore/ #06-984 NQO1 1:1000 Cabra Pc Abcam/ #ab2346 NRF2 1:1000 Conejo Pc No comercial p38 1:2000 Conejo Pc Santa Cruz/ #sc-535 p-p38 1:1000 Conejo Pc Cell Signaling/ #9211 Vinculina 1:15000 Ratón Mc Santa Cruz/ #sc-73614 IgG de cabra 1:5000 Conejo Bio-Rad Laboratories/ #1721034 IgG de conejo 1:3000 Cabra Bio-Rad Laboratories/ #1706515 IgG de ratón 1:3000 Cabra Bio-Rad Laboratories/ #1706516 Membranes were then incubated with a peroxidase‐conjugated secondary antibody for 1 h at room temperature, and bands were visualized using Clarity™ Western ECL Substrate (Bio‐Rad) using an ImageQuant LAS4000 mini digital camera (GE Healthcare Bio‐Sciences, Pittsburgh, PA, USA) and densities were quantified using Image Quant TL software. ENZYMATIC ACTIVITY DATA: The measurement of the enzyme activity of G6PD, 6PGD and SOD was performed in mitochondrial or cytosolic extracts. Samples were manually homogenized in a buffer extraction. The lysates were centrifuged to obtain the nuclear fraction (precipitated). The supernatants were centrifuged to obtain the cytosolic (supernatant) fraction. The mitochondrial fraction was obtained by homogenizing the precipitated. The homogenized precipitate is Incubated in the extraction buffer for 30 minutes on ice and centrifuged at 13,000 rpm for 10 minutes at 4ºC. Concentration protein of each of the cell fractions was determined using the DCTM Protein kit Assay Kit II (Bio-Rad Laboratories). The activity of the enzymes G6PD and 6PGD was determined in the cytosolic fractions, as reported (White et al., 2017). The enzymatic activity of SOD was determined in the cytosolic and using the EpiQuikTM Superoxide Dismutase Activity/Inhibition Assay Kit (EpiGentek). Optical density was measured with the VERSAmaxTM spectrophotometer Tunable Microplate Reader using SOFTmax® Pro 3.0 Software