Figure 2

Biophysical and structural characterization of neurotoxic Aβ assembly. (Upper half of panel A) Representative calcein AM/PI stainings of NGF-treated PC12 (PC12N) cells treated at 37°C for 48 h with: TBS alone; 0-h preincubated Aβ1-42 (0 h); 2-h preincubated Aβ1-42 (2 h); 540,000 g supernatant obtained from 2 h (2 h sup); 4-h preincubated Aβ1-42 (4 h); 540,000 g supernatant obtained from 2 h (4 h sup). Green staining for viable cells versus red staining for dead cells. Resultant cell viability for each treatment is shown in lower half of panel A. Experimental results were analyzed by one-way ANOVA, followed by Tukey's test for posthoc analysis: statistical significance compared with TBS alone (*p<0.0001). Scale bar = 50 μm. (B) The seed-free Aβ1-42 (25 μM) was subjected to a series of membrane ultrafiltration steps with molecular cutoffs at 3, 10, 30, and 100 kDa. The resultant four filtrates and one retentate were designated as Fr. 1 (<3 kDa), Fr. 2 (3-10 kDa), Fr. 3 (10-30 kDa), and Fr. 4 (30-100 kDa), and final retentate Fr. 5 (>100 kDa). The upper half of panel B shows the representative calcein AM/PI stainings of NGF-treated PC12 (PC12N) cells incubated at 37°C for 48 h with seed-free unfractionated Aβ1-42 or five fractions (25 μM each). Resultant cell viability for each treatment is shown in the lower half of panel B. Experimental results were analyzed by one-way ANOVA, followed by Dannett's test for posthoc analysis: statistical significance compared with TBS alone (*p<0.001). (C) Dot blot analysis of five fractions (Frs. 1-5). The blots were reacted with A11, 1A9, 2C3, and 4G8. (D) Amplitude AFM images (2 μm × 2 μm) of four fractions (Frs. 2-5). All AFM images were taken on a mica surface.