Fig 1: Characterization of tau fibrils extracted from human AD subjects.A, immunoblots for total tau (HT7) and pTau-Thr231 (AT180) of the tau extracts used in this study (samples were as follows: C: control, A: brain A, B: brain B, AB: 1:1 mixture of brain A and B extracts, D: brain D, and Dconc: 10× concentrated brain D). B, overview of total protein and total tau concentration in each extract. Total protein concentration was evaluated by measuring absorption at 280 nm on a spectrophotometer. Total tau concentration was calculated by comparing densitometric quantifications of total tau (HT7) immunoblots to a dilution curve of recombinant tau (2N4R isoform). C, immunoblots for total tau (HT7) and pTau-Thr231 (AT180) for samples from brain A, B, and a 1:1 mixture of brain A and B (AB) to assess the tau isoform distribution and phosphorylation over a 60-h time course with measurements taken every 12 h. D, tau extracts were diluted in PBS to a concentration of 5 μg/ml, and 5 × 5 μm images were taken by atomic force microscopy (the scale bar represents 0.5 μm). Fibril length was quantified using Gwyddion. N(A) = 219 fibrils from two images, N(B) = 260 fibrils from three images, N(AB) = 494 fibrils from three images, N(D) = 116 fibrils from two images, and N(Dconc) = 225 fibrils from two images. E, primary mouse neurons were seeded at 50,000 to 75,000 density, treated with 0.25% (v/v) human tau extracts at DIV 7 and incubated until DIV 21 (the scale bar represents 50 μm). Cells were fixed in ice-cold methanol and stained for MAP2 and mouse tau (T49). Tau seeding was measured by quantifying the percent area occupied by aggregated mouse tau within MAP2-positive area using ImageJ. Statistics: Brown–Forsythe ANOVA test (F = 43.85; p < 0.0001) with Dunnett's multiple comparisons test to compare all groups to control-treated cells. N = 8 images per condition. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.005, and ∗∗∗∗p < 0.0001. AD, Alzheimer's disease; DIV, days in vitro.