Journal: Frontiers in Molecular Biosciences

Article Title: Accumulated ROS Activates HIF-1α-Induced Glycolysis and Exerts a Protective Effect on Sensory Hair Cells Against Noise-Induced Damage

doi: 10.3389/fmolb.2021.806650

Figure Lengend Snippet: Glucose metabolic characteristics in t-BHP-treated HEI-OC1 cells. (A) The mitochondrial content of HEI-OC1 cells after treatment with 80 μM t-BHP for 4 h in comparison with controls. Texas Red fluorescence arising from Mito-tracker was measured by flow cytometry. (B) ATP production of t-BHP-treated HEI-OC1 cells relative to controls, as determined by fluorometric analysis. ATP production was normalized to total protein concentration. (C) 2-NBDG uptake by HEI-OC1 cells after treatment with 80 μM of t-BHP for 4 h in comparison with controls. FITC fluorescence derived from 2-NBDG was measured by flow cytometry. (D) Lactate release from t-BHP-treated HEI-OC1 cells compared to controls as determined by colorimetric analysis. Lactate levels were normalized to total protein concentration. (E) The enzymic activity of LDH in t-BHP-treated HEI-OC1 cells relative to controls, as determined by colorimetric analysis. (F) The enzymic activity of LDH in mitochondrial isolation of t-BHP-treated HEI-OC1 cells relative to controls, as determined by colorimetric analysis. (G) HEI-OC1 cells were seeded in a XFe96 cell culture plate. Next, we added 1.5 μM oligomycin, 1 μM FCCP, and 0.5 μM rotenone/antimycin A and monitored the OCR continuously. (H) HEI-OC1 cells were seeded in XFe96 cell culture plates. We then added 100 mM glucose, 10 μM oligomycin, and 500 mM 2-DG and monitored ECAR continuously. (I) The illustration of the typical OCR results. (J) A comparison of basal respiration, mitochondrial ATP production, maximal respiration, and non-mitochondrial oxygen consumption in t-BHP-treated HEI-OC1 cells relative to controls. (K) The illustration of the typical ECAR results. (L) A comparison of non-glycolytic acidification, basal glycolysis, glycolytic capacity, and glycolytic reserve, in t-BHP-treated HEI-OC1 cells relative to controls. 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).

Article Snippet: 2 × 10 5 indicated HEI-OC1 cells were seeded per well in 6-well plates, grown to 80% confluence in a 33°C 10% CO 2 humidified incubator and then treated or without 80 μM t-BHP for 4 h. Cells were incubated with sugar-free DMEM medium with 100 μM fluorescent glucose analog 2-NBDG (MCE, China) for 2 h in an incubator.

Techniques: Comparison, Fluorescence, Flow Cytometry, Protein Concentration, Derivative Assay, Activity Assay, Isolation, Cell Culture

Journal: Frontiers in Molecular Biosciences

Article Title: Accumulated ROS Activates HIF-1α-Induced Glycolysis and Exerts a Protective Effect on Sensory Hair Cells Against Noise-Induced Damage

doi: 10.3389/fmolb.2021.806650

Figure Lengend Snippet: HIF-1α-induced glycolysis in response to oxidative stress in HEI-OC1 cells. (A) Western blots of HIF-1a expression in HEI-OC1 cells treated with or without t-BHP. β-Tubulin was used as an internal reference. (B) RT-qPCR analysis for the expression levels of Hif1a in t-BHP-treated HEI-OC1 cells relative to controls. Tubb3 was used as an internal reference. (C) Immunocytofluorescence staining of HIF-1a (green); nuclei were stained blue with DAPI. Scale bar = 10 μm. (D) siRNA was used to knock down Hif1a expression levels in HEI-OC1 cells. The knockdown of Hif1a was confirmed by western blotting. β-Tubulin was used as a loading control. (E) The viabilities of HEI-OC1 cells transfected with siNC and si Hif1a and treated with or without 80 μM t-BHP were determined by CCK8 assays. (F) The ATP production by HEI-OC1 cells transfected with siNC and si Hif1a and treated with or without 80 μM t-BHP was determined by fluorometric analysis. ATP production was normalized to total protein concentration. (G) 2-NBDG uptake by HEI-OC1 cells transfected with siNC and si Hif1a and treated with or without 80 μM t-BHP was determined by flow cytometry. (H) Lactate release by HEI-OC1 cells transfected with siNC and si Hif1a and treated with or without 80 μM t-BHP was determined by colorimetric analysis. Lactate levels were normalized to total protein concentration. (I) RT-qPCR analysis of the expression levels of Glut1 and glycolytic enzymes in HEI-OC1 cells transfected with siNC and si Hif1a and treated with or without 80 μM t-BHP. Tubb3 was used as an internal reference. (J) Western blots of GLUT1 and the expression levels of glycolytic enzymes in HEI-OC1 cells transfected with siNC and siHif1a and treated with or without 80 μM t-BHP. β-Tubulin was used as an internal reference. Data are represented as mean ± SEM. Bar charts were compared by Student’s t-test or ANOVA (ns = not significant, * p < 0.05, ** p < 0.01, and *** p < 0.001).

Article Snippet: 2 × 10 5 indicated HEI-OC1 cells were seeded per well in 6-well plates, grown to 80% confluence in a 33°C 10% CO 2 humidified incubator and then treated or without 80 μM t-BHP for 4 h. Cells were incubated with sugar-free DMEM medium with 100 μM fluorescent glucose analog 2-NBDG (MCE, China) for 2 h in an incubator.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Staining, Knockdown, Control, Transfection, Protein Concentration, Flow Cytometry