Fig. 6

HMGB1/RAGE axis blocks the late stages of mitophagy flux in stressed microglia. LC3 dots were visualized under fluorescent confocal microscope and quantified following mRFP-GFP-tandem fluorescent LC3 adeno-associated virus transfected to the RVLM in vivo (a, b) (scale bar = 50 μm) and ex vivo (c, d) (scale bar = 2 μm). e–g Whole cell lysates were collected at the indicated time points and analyzed by immunoblotting for LC3II, p62, and β-actin (loading control). The levels of the p62 and LC3-II were increased in the stressed microglia. h Microglia stained with Lyso-Tracker-red and MitoTracker Green to label lysosomes and mitochondria colocalization (scale bar = 2 μm). i The levels of colocalization of MitoTracker and Lyso-Tracker were assessed by using the Pearson coefficient. j Electron micrograph showed few typical autophagosomes in normal cytoplasm of the control microglia. An increased number of mitophagy vesicles were observed in stressed microglia. Red arrows indicate the representative mitophagy, which was visualized as mitochondria-containing autophagosome (scale bar = 0.5 μm). Data are presented as mean ± SEM. n = 6, *P < 0.05, ANOVA LSD test