The PROMO database (alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3/) was used to predict the association between UCHL1 promoter and Sp1.

Murine cochlea hair cells (HEI-OC1; cat. no. BFN60808695), obtained from BLUEFBIO, were cultured in DMEM supplemented with 10% FBS (both Shanghai ExCell Biology, Inc.) at 33˚C with 10% CO₂. To establish an in vitro ARHL model, HEI-OC1 cells were exposed to 1 mM hydrogen peroxide (H₂O₂) for 2 h at 33˚C, as previously described (18,19).

Total RNA was extracted from HEI-OC1 cells (6-well plates at a density of 6x10⁴ cells per well) using TRIzol^® reagent (Invitrogen) and cDNA was synthesized using the AffinityScript cDNA synthesis kit (Agilent Technologies) according to the manufacturer's instructions. qPCR was performed on a LightCycler 480 PCR instrument (Roche Diagnostics) using the AceQ^® qPCR SYBR Green Master Mix (Vazyme Biotechnology Co. Ltd.). The following thermocycling conditions were used for qPCR: Pre-denaturation at 95˚C for 1 min, followed by 40 cycles of denaturation at 95˚C for 15 sec, annealing at 60˚C for 40 sec and extension at 72˚C for 15 sec. The changes in gene expression levels were assessed using the 2^-ΔΔCq method (20). The following primer pairs were used for qPCR: UCHL1, forward, 5'-AGGGACAGGAAGTTAGCCCTA-3' and reverse, 5'-AGCTTCTCCGTTTCAGACAGA-3'; Sp1 forward, 5'-CCTGGCATCCCACCAGAGTA-3' and reverse, 5'-GTGCAAGGAGCTGATCCCAA-3' and β-actin forward, 5'-GTTGGAGCAAACATCCCCCA-3' and reverse, 5'-CGCGACCATCCTCCTCTTAG-3'.

Total protein was extracted from HEI-OC1 cells (1x10⁶ cells) using RIPA lysis buffer (Shanghai Yisheng Biotechnology Co., Ltd.) and the protein concentration was determined using a BCA protein assay kit (Beijing Solarbio Science & Technology Co., Ltd.). The proteins were transferred onto PVDF membranes (30 µg/lane) following separation by SDS-PAGE on a 10% gel. Following blocking with 5% skimmed milk at room temperature for 1 h, membranes were first incubated with primary antibodies at 4˚C overnight and then with a goat anti-rabbit horseradish peroxidase-conjugated secondary antibody (cat. no. ab205718; 1:2,000; Abcam) for 1 h at room temperature. The ECL Western Blot kit (Jiangsu CoWin Biotech Co., Ltd.) was used to develop the immunoreactive signals and protein band intensity was calculated using Image-Pro Plus software (version 6.0; Media Cybernetics, Inc.). The primary antibodies were as follows: Anti-UCHL1 (cat. no. ab108986; 1:1,000; Abcam), anti-B cell lymphoma-2 (Bcl-2; cat. no. ab182858; 1:2,000; Abcam), anti-Bcl-2 associated X (Bax; cat. no. ab32503; 1:1,000; Abcam), anti-p16 (cat. no. ab51243; 1:1,000; Abcam), anti-p21 (cat. no. ab109199; 1:1,000; Abcam), anti-Sp1 (cat. no. ab227383; 1:1,000; Abcam), anti-phosphorylated (p)-NF-κB p65 (cat. no. ab76302; 1:1,000; Abcam), anti-NF-κB p65 (cat. no. ab32536; 1:1,000; Abcam) and anti-β-actin (cat. no. ab8227; 1:1,000; Abcam).

The recombinant plasmids pcDNA3.1-UCHL1 (Oe-UCHL1) and pcDNA3.1-Sp1 (Oe-Sp1), as well as the empty vector pcDNA3.1 negative control (Oe-NC), were obtained from Sangon Biotech Co., Ltd. HEI-OC1 cells seeded into six-well plates at a density of 5x10⁵ cells/well were transfected with 5 µg plasmids using Lipofectamine^® 2000 (Life Technologies; Thermo Fisher Scientific, Inc.) at 37˚C for 48 h, according to the manufacturer's recommendation. The subsequent experiments were performed at 48 h following cell transfection.

To assess cell viability, a CCK-8 kit (Abnova) was used according to the manufacturer's instructions. Briefly, HEI-OC1 cells were seeded into 96-well plates at a density of 5x10³ cells/well and treated with 10 µl CCK-8 solution at 37˚C for 2 h. Subsequently, optical density (OD) at a wavelength of 450 nm was measured using a microplate reader (Infinite M200; Tecan Group, Ltd.).

HEI-OC1 cells were seeded into 96-well plates (5x10³ cells/well). Following cell lysis in 300 µl lysis buffer (0.1% SDS, 0.5% Triton X-100, 20 mM Tris-HCl, pH 8.1), protein concentration was measured using a BCA protein assay kit (Bio Basic, Inc.). Assay kits for SOD (cat. no. A001-1-2) and GSH-Px (cat. no. A005-1-2) were obtained from Nanjing Jiancheng Bioengineering Institute and used according to the manufacturer's instructions. The OD values at a wavelength of 450 nm were determined using a microplate reader (Infinite M200; Tecan Group, Ltd.). To measure ROS accumulation, HEI-OC1 cells seeded into 24-well plates were treated with 10 µΜ dichloro-dihydro-fluorescein diacetate (MilliporeSigma) at 37˚C for 30 min in the dark followed by washing with serum-free DMEM three times. ROS generation was assessed by flow cytometry (Merck KGaA) using FACSAria (BD Biosciences) using the corresponding kit (cat. no. ab113851; Abcam) according to the manufacturer's instructions. The data were viewed in FlowJo software (version 10; FlowJo, LLC).

HEI-OC1 cells were fixed with 4% paraformaldehyde for 30 min at room temperature and were then treated with 0.1% Triton X-100 for 5 min at room temperature. TUNEL assay was performed using TUNEL reagent (cat. no. MK1013; Wuhan Boster Biological Technology, Ltd.) for 60 min at 37˚C according to the manufacturer's instructions. Cell nuclei were labeled with 10 mg/ml DAPI for 5 min at room temperature in the dark and images were captured from four random fields under a fluorescence microscope (LSM800; Carl Zeiss AG) after Antifade Mounting Medium (Beyotime Institute of Biotechnology) was added to the sections.

Briefly, HEI-OC1 cells were seeded in six-well plates at a density of 5x104 cell/well. HEI-OC1 cells at ~80% confluency plated into 6-well plates were treated with 4% formaldehyde for 15 min at room temperature. Following washing with PBS, cells were incubated with SA-β-gal staining solution (cat. no. K320-250; BioVision, Inc.) overnight at 37˚C without CO₂. Finally, stained cells from 3 random fields of view were observed under a light microscope (Carl Zeiss AG).

Wild-type (WT; 5'-CCCGCCCCG-3') or mutant (MUT; 5'-CAAAAAAAC-3') UCHL1 promoter were cloned into the pGL3 Basic vector (Promega Corporation). Cells (5x10⁵) were seeded in 24-well plates for 24 h at 37˚C and were transfected with these plasmids as well as with Oe-Sp1 and Oe-NC using Lipofectamine^® 2000 (Life Technologies; Thermo Fisher Scientific, Inc.) at 37˚C. After 48 h transfection, the luciferase activity was measured using the Dual-Luciferase Reporter Gene Assay kit (Shanghai Qcbio Science & Technologies Co., Ltd.) according to the manufacturer's instructions. The relative luciferase activity was normalized to that of Renilla luciferase.

ChIP assay was performed using the ChIP kit (Abcam). Briefly, 1% formaldehyde was added to HEI-OC1 cells for 10 min at room temperature. The fixed cells were washed twice with phosphate-buffered saline and were lysed using a lysis buffer (0.1% SDS, 0.5% Triton X-100, 20 mM Tris-HCl, pH 8.1) that contained 150 mM NaCl and a protease inhibitor, after which chromatin fragments were obtained using sonication using a 10 sec on and 10 sec off mode for 12 cycles at 4˚C. Following centrifugation at 13,000 x g for 10 min at 4˚C, the DNA fragments were incubated with antibodies against Sp1 (cat. no. ab227383; 1:200: Abcam) or IgG (cat. no. ab6702; 1:40; Abcam) for 2 h at 4˚C. The abundance of Sp1 on the UCHL1 promoter was measured by PCR as aforementioned. The sequence of oligonucleotides flagging the Sp1 binding site in the UCHL1 promoter was 5'-CCCGCCCCC-3'.

All statistical analyses were performed using GraphPad Prism version 8 software (GraphPad Software, Inc.). Continuous variables are expressed as the mean ± standard deviation from three independent experiments. The differences between two groups were compared using unpaired Student's t test, while those between multiple groups were by one-way ANOVA followed by Tukey's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

To evaluate the role of UCHL1 in H₂O₂-induced HEI-OC1 cells, expression levels of UCHL1 were measured. RT-qPCR and western blot analysis showed that UCHL1 was downregulated in H₂O₂-treated HEI-OC1 cells (Fig. 1A and B). To determine the effect of UCHL1 on H₂O₂-treated HEI-OC1 cells, UCHL1 was overexpressed following cell transduction with Oe-UCHL1 plasmid. The overexpression efficiency was verified via RT-qPCR and western blot analysis (Fig. 1C and D). Furthermore, CCK-8 assay demonstrated that treatment with H₂O₂ diminished HEI-OC1 cell viability. However, UCHL1 overexpression increased the viability of HEI-OC1 cells exposed to H₂O₂. These findings indicated that UCHL1 protected HEI-OC1 cells from H₂O₂-triggered cell injury.

Contents of the oxidative stress-related markers SOD, GSH-Px and ROS were measured using the corresponding kits. The results revealed that exposure to H₂O₂ decreased SOD and GSH-Px levels but increased ROS activity. However, UCHL1 overexpression increased SOD and GSH-Px levels and attenuated ROS activity in H₂O₂-treated HEI-OC1 cells (Fig. 2A). Furthermore, TUNEL assay showed that UCHL1 ameliorated H₂O₂-induced HEI-OC1 cell apoptosis (Fig. 2B and C). In addition, western blot analysis revealed that cell exposure to H₂O₂ downregulated Bcl-2 and upregulated Bax, which were reversed by UCHL1 overexpression (Fig. 2D). Overall, these results suggested that UCHL1 exerted an inhibitory effect on H₂O₂-triggered oxidative stress and apoptosis in HEI-OC1 cells.

SA-β-gal staining illustrated that the increased number of SA-β-gal-positive H₂O₂-treated HEI-OC1 cells was decreased following UCHL1 overexpression (Fig. 3A). In addition, UCHL1 overexpression suppressed the H₂O₂-mediated enhanced expression levels of p16 and p21 (Fig. 3B). These results indicated that the H₂O₂-mediated HEI-OC1 cell senescence was attenuated by UCHL1 overexpression.

The PROMO database revealed that the UCHL1 promoter interacted with the Sp1 transcription factor. RT-qPCR and western blot analysis revealed that Sp1 was upregulated in H₂O₂-induced HEI-OC1 cells (Fig. 4A and B). Following Sp1 overexpression by cell transduction with Oe-Sp1 plasmid (Fig. 4C and D), luciferase reporter assay showed that Sp1 overexpression diminished the luciferase activity of UCHL1-WT compared with UCHL1-MUT (Fig. 4E). ChIP assay showed that UCHL1 was precipitated following incubation of nuclear extracts with Sp1 antibody (Fig. 4F). Additionally, Sp1 overexpression decreased expression levels of UCHL1 (Fig. 4G and H). Collectively, the aforementioned findings demonstrated that Sp1 was a transcriptional inhibitor of UCHL1.

To uncover the association between Sp1 and UCHL1 in H₂O₂-treated HEI-OC1 cells, functional experiments were performed in H₂O₂-induced HEI-OC1 cells co-transduced with Oe-UCHL1 and Oe-Sp1 plasmids. CCK-8 assay revealed that UCHL1 restored the suppressed viability of H₂O₂-induced HEI-OC1 cells. However, this effect was reversed by Sp1 overexpression (Fig. 5A). The enhanced SOD and GSH-Px activity, as well as the reduced ROS levels mediated by UCHL1 overexpression in H₂O₂-induced HEI-OC1 cells, were restored following Sp1 overexpression (Fig. 5B). In addition, the attenuated H₂O₂-induced HEI-OC1 cell apoptosis mediated by UCHL1 overexpression was further increased in cells co-transfected with Oe-Sp1 plasmid (Fig. 5C). This was further verified by western blot analysis, showing that Sp1 overexpression abrogated the enhanced Bcl-2 and decreased Bax expression levels in H₂O₂-exposed HEI-OC1 cells co-transduced with Oe-UCHL1 plasmid (Fig. 5D). Additionally, SA-β-gal staining and western blot analysis demonstrated that the decreased number of SA-β-gal-positive cells, as well as p61 and p21 downregulation triggered by UCHL1 overexpression in H₂O₂-induced HEI-OC1 cells, were reversed by Sp1 overexpression (Fig. 5E and F). Notably, UCHL1 overexpression restored the H₂O₂-induced p-NF-κB p65 upregulation, which was further abolished by Sp1 elevation (Fig. 5G). Taken together, these findings indicated that UCHL1, negatively regulated by Sp1, promoted H₂O₂-mediated cell injury, oxidative stress, apoptosis and senescence, and modulated NF-κB signaling in HEI-OC1 cells.