Fig. 3: TRPV1 activation reversed ApoE4-induced neuronal autophagy impairment and lipid droplet accumulation in vitro.

a, b Western blot and quantification of phospho-mTOR (Ser2448), phospho-Akt (Thr308), and phospho-GSK3β in SH-sy5y cells treated with 150 nM recombinant ApoE and 100 μM PA for 24 h. c Representative immunofluorescent images and quantification of BODIPY⁺LC3⁺ puncta in primary neurons. d Quantification of cellular MMP in primary neurons stained with JC-1. e Quantification of cellular ROS production in primary neurons stained with DCFH-DA. f–i Representative immunofluorescent images and quantification of Parkin and MitoTracker Red (f, g) or LC3 and MitoTracker Red (h, i). j Mitochondrial stress test of 150 nM ApoE3- and ApoE4-treated neurons for 24 h with pretreatment with 10 μM capsaicin; basal respiration and maximal respiration are shown. k Confocal images of MAP2⁺ (neurite) cells treated with 150 nM ApoE3 and ApoE4 for 24 h. Quantification of the mean neurite length of MAP2⁺ cells from confocal images. Experiments on primary neurons were performed three times in technical triplicates. Statistical tests: one-way ANOVA followed by Tukey’s post hoc test (b–e, g, i, j, k). Data represent the mean ± s.e.m. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bars, 25 μm (c), 20 μm (f, h), 100 μm (k).