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. 2022 Jan 13;119(3):e2115082119. doi: 10.1073/pnas.2115082119

Fig. 3.

Fig. 3.

KARI 201 ameliorates defective autophagy degradation by regulating lysosomal biogenesis in neurons. (A) Autophagic flux assay. Mouse neurons were cultured in medium with or without ASM (10 µM) or KARI 201 (10 µM) under complete medium or starvation conditions. The LC3-II levels were examined by Western blotting. The accumulation of p62 was assessed with ASM (10 µM), KARI 201 (10 µM), NH4Cl (20 mM), or starvation (n = 4 per group). (B) The levels of TFEB and Lamp1 in mouse neurons (n = 4 per group). (C) Immunocytochemistry of Lamp1 in ASM-treated cells with or without KARI 201 (n = 6 per group). (Scale bars, 5 μm.) (D) Western blot analysis for nuclear localization of TFEB in ASM-treated cells with or without KARI 201 (n = 4 per group). (E) qRT-PCR analysis of TFEB-target gene expression in each group (n = 4 per group). (F and G) Western blot analyses and quantification for LC3, Beclin-1, p62, cathepsin D, Lamp1, and TFEB in cortex (F) and hippocampus (G) in WT and APP/PS1 mice treated with PBS or KARI 201 (n = 6 mice per group). (H) The expression of TFEB-target genes in each group (n = 4 mice per group). One-way ANOVA, Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001. All error bars indicate SEM. All data analysis was done in 9-mo-old mice.