Figure 3.

V-ATPase ATP6V0C subunit is functionally associated with Aβ toxicity. (A) Silver staining of V-ATPase complexes purified from the brains of mice through a high-affinity purification strategy involving SidK. (B) Eight μg purified V-ATPases were incubated with 3 μg Aβ in PBS containing 1% Triton-X 100 for 3 h, and the affinity of V-ATPase subunits (ATP6V0C, ATP6V1A, and ATP6V1B2) for Aβ were analyzed by co-IP with anti-Aβ (4G8) antibody. (C) ATP6V0C-knockdown HT22 stable cells were established using a CRISPR/Cas9 system and the endogenous ATP6V0C level was analyzed by western blotting; HT22 control (left), sgRNA ATP6V0C #14 (middle), and #19 (right) stable cells. (D) HT22 control, sgRNA ATP6V0C #14, and #19 stable cells were transfected with EGFP or EGFP-ATP6V0C for 24 h and further treated with Aβ for 48 h. Cytotoxicity was analyzed by PI staining (n = 3, cells > 150 for each trial), two-way ANOVA. (E) HT22 control and sgRNA ATP6V0C #14 cells were treated with 5 μM Aβ for 24 h. Proteolysis activity was analyzed by measuring DQ-red-BSA:Cascade Blue-dextran fluorescence ratio (n = 3, cells > 35 for each trial), two-way ANOVA. Scale bar: 10 μm. (F) HT22 control and sgRNA ATP6V0C #14 cells stably expressing pHLARE were reconstituted with ATP6V0C gene and then treated with 5 μM Aβ for 24 h. The pH was measured by calibrating sfGFP:mCherry fluorescence ratio (n = 5, each dot presents the average pH of cells > 300 per trial), two-way ANOVA. (G) Schematic representation of the structure of the ATP6V0C subunit of V-ATPase. (H, I) HT22 cells were co-transfected with pEGFP, and pcDNA-HA (Control), indicated HA-ATP6V0C WT or mutants and further treated with 5 μM Aβ for 48 h. The apoptotic cell death was analyzed by PI staining. Two-way ANOVA (H, n = 3, cells > 100 for each trial; I, n = 4, cells > 100 for each trial) (upper). Expression of HA-ATP6V0C WT or mutants were analyzed by western blotting (lower).