Figure 2
Accumulation of AVs under hypoxic stress. (a) Neuro-2a cells were submitted to hypoxia and/or high glucose for 48 h, and the protein levels of P62 and LC3B-II were measured by immunoblots, showing a time-dependent increase in levels of LC3-II and P62 under hypoxia compared with those under normoxia. In contrast, high glucose had no significant effect on their expression. (b) The ImageJ densitometric analysis of P62 and LC3B-II from the immunoblots is shown. “C” represented control conditions (25 mM glucose), “G” represented high glucose conditions (75 mM glucose). The results are expressed as the mean ± SEM from three independent experiments. Two-way ANOVA, **p < 0.01, Hypoxia vs Normoxia. (c) Immunofluorescent assay for the endogenous distribution and localization of LC3B in Neuro-2a cells. Neuro-2a cells were submitted to hypoxia and/or high glucose for 48 h and then fixed. LC3B was visualized as a green signal, and the blue sections are nuclei stained by DAPI. Scale bar, 20 μm. (d) Histogram shows the number of AVs per cell (LC3B-positive puncta) in Neuro-2a cells. The results are expressed as the mean ± SEM from three independent experiments with nine microscopy fields for each experiment. Two-way ANOVA, **p < 0.01, Hypoxia vs Normoxia. Full-length blots are presented in Supplementary Fig. S2.
