Fig 2: Oxidative stress drives glycolysis in HEI-OC1 cells. (A) Western blots of GLUT1 and the expression levels of glycolytic enzymes in HEI-OC1 cells treated with or without t-BHP. ß-Tubulin was used as an internal reference. (B) RT-qPCR analysis of the expression levels of Glut1 and glycolytic enzymes in t-BHP-treated HEI-OC1 cells relative to controls. Tubb3 was used as an internal reference. (C) Immunocytofluorescence staining of GLUT1 and LDHA, the nuclei were stained blue with DAPI. Scale bar = 10 µm. (D) Cell viability was measured by CCK8 assays on treatment with or without 50 µM 3-BrPA or 2 µM AZ-33 for 48 h. HEI-OC1 cells cultured without 3-BrPA and AZ-33 were used as a baseline. (E) ATP production of 50 µM 3-BrPA or 2 µM AZ-33-treated HEI-OC1 cells relative to controls, as determined by fluorometric analysis. ATP production was normalized to total protein concentration. (F) Effect of glucose deprivation or exogenous lactate supplementation on the survival of HEI-OC1 cells. Different concentrations of glucose and lactate were added to the medium for 48 h. HEI-OC1 cells cultured in normal complete DMEM medium were used as a baseline. Data are represented as mean ± SEM. Bar charts were compared by the Student’s t-test (ns = not significant, *p < 0.05, **p < 0.01, and ***p < 0.001).

Fig 3: The activated oxidative stress-HIF-1a-glycolysis axis in NIHL mice. (A) Representative confocal images of the organ of Corti immunolabeled for Myosin VIIa (red) and 4-HNE (green), nuclei were stained blue by DAPI. Scale bar = 20 µm. Asterisks indicate the absence of outer hair cells. (B) Representative confocal images of the organ of Corti immunolabeled for Myosin VIIa (red) and HIF-1a, GLUT1, or LDHA (green), nuclei were stained blue by DAPI Scale bar = 20 µm.

Fig 4: Two-dimensional (A) and 3D (B) patternsillustrating the proposed binding mode of the compound 5c in the LDHA binding pocket (PDB code: 4ZVV). Hydrogen bonding interactions with the protein are shown as dashed lines. Green color reflects a hydrophobic area, pink color reflects a high polar area, and blue color reflects a mild polar area.

Fig 5: Two-dimensional (A) and 3D (B) patternsillustrating the proposed binding mode of the co-crystallized ligand GNE-140 in the LDHA binding pocket (PDB code: 4ZVV). Hydrogen bonding interactions with the protein are shown as dashed lines. Green color reflects a hydrophobic area, pink color reflects a high polar area, and blue color reflects a mild polar area.