Figure 2.

Accumulation of Aβ leads to proteolytic dysfunction in endolysosomes. (A, B) HT22 cells were treated with 5 μM Aβ in the absence or presence of 100 μM MDC for 48 h (A) or 24 h (B). Cytotoxicity was analyzed by PI and calcein-AM staining (n = 3, cells > 400 for each trial) (A). Proteolysis activity was analyzed by measuring DQ-red-BSA:Cascade Blue-dextran fluorescence ratio (n = 3, cells > 50 for each trial) (B). Two-way ANOVA. (C) HT22 cells were co-treated with 5 μM DyLight 594-labeled Aβ, 10 μg/ml DQ-green-BSA and 10 μg/ml Cascade Blue-dextran for 24 h (upper). Pixel intensities in dual-channel images were analyzed and displayed as a scatter plot (lower). (D) Microsomes purified from mouse brains were incubated with 1 μM Baf.A1 or liposomes pre-loaded with 2.5 μM Aβ or 3 μM BSA (shown in Fig. S2C). The amount of phosphate released from the microsomes was determined by the malachite green method (n = 4), two-way ANOVA. (B, C) Scale bar: 10 μm.