Figure 2

ROS generation, mitophagy level, and PHB2 expression in HG- or AGE-treated HT-22 cells. (a–d) Cell viability of HT-22 cells treated with different concentrations of glucose and mannitol (a, b) or AGEs (c, d) for 24 or 48 h, as measured by CCK-8 assay; ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs 25 mM glucose or 0 μg/mL AGE group (n = 3). (e–h) Representative fluorescence images of Mito-Tracker (green) and Lyso-Tracker (red) and their relative co-locations in HT-22 cells treated with HG (e, f) or AGEs (g, h) for 48 h (scale bar = 20 μm). (i–p) Representative WB images (i, m) and quantitative analysis of ATG5/Beclin 1 (j, o), LC3II/LC3I (k, n), and TOM20 (l, p) proteins in HT-22 cells treated with different concentrations of glucose (i–l) or AGEs (m–p); ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs 25 mM glucose or 0 μg/mL AGE group (n = 3). (q, r) Representative fluorescence images (q) and relative ROS levels (r) in HT-22 cells treated with glucose (100 mM) or AGEs (400 μg/mL) for 48 h (scale bar = 20 μm); ^**P < 0.01 and ^***P < 0.001 vs CON group. (s, t) RT-PCR analysis of PHB2 expression in the HT-22 cells treated with different concentrations of glucose (s) or AGEs (t). β-actin was used as the internal control; ^*P < 0.05 and ^**P < 0.01 vs 25 mM glucose or 0 μg/mL AGE group. The values are shown as the means ± SD (n = 3).