Fig. 2: Clusterin enhances the seeding potency of FLTau aggregates.

a Effect of Clu on the kinetics of aggregation of full-length (FL) Tau (10 μM) monitored by ThT fluorescence. FLTau/Clu molar ratio was 1:1. Averages ± SEM (n = 5 independent experiments). arb.units, arbitrary units. b Formation of seeds in FLTau aggregation reactions without (black) or with Clu (10 μM, red) as described in (a). Reporter cells were transfected with aggregation reactions containing 183 ng (solid lines) or 4.6 ng (dashed lines) FLTau and 205 ng (solid lines) or 5.1 ng (dashed lines) Clu, respectively (molar ratio Clu:FLTau 1:1). Averages ± SEM (n = 3 independent experiments). c Representative fluorescence microscopy images of TauRD-YT cells seeded with FLTau aggregation reactions (183 ng FLTau) after reaching the plateau of aggregation (10 days, (a)). TauRD-YFP and DAPI nuclear staining signals are shown in yellow and blue, respectively. Scale bars, 20 μm. d Fold change of seeding potency of FLTau aggregation reactions containing Clu (red) compared to control reactions without Clu (gray). Bar graphs represent the average slope ± SEM (n = 3 independent experiments) from the linear regression analyses described in Supplementary Fig. 5a. *p < 0.05 (p = 0.0106) by two-tailed Student’s t-test. Lipofectamine was used as a transfection reagent. e, f Seeded aggregates of FLTau-YT contain phospho-Tau. (n = 3 independent experiments) e Fluorescence microscopy images of FLTau-YT cells seeded with TauRD aggregates. FLTau-YFP and immunostaining of phospho-Tau (pTau, AT8 antibody) are shown in yellow and red, respectively. The AT8 antibody recognizes Tau phosphorylation at both serine 202 and threonine 205 and is widely used to detect Tau paired helical fibrils^36,49. DAPI nuclear staining signal is additionally shown in blue in the merge. Arrowheads indicate aggregates. The small arrow indicates a cell without aggregates. Scale bar, 10 μm. f Representative immunoblot analysis showing Tau (Tau/Repeat Domain antibody) and phospho-Tau (pTau, AT8 antibody) in FLTau-YT cell lysates from cells treated with or without TauRD seeds. Molecular weight (MW) standards are indicated. g Clu enhances the seeding potency of FLTau aggregates formed in FLTau-YT cells. Whole-cell lysates of FRET-positive (pos.) FLTau-YT cells were incubated without or with Clu (molar ratios FLTau-YT:Clu 1:1 and 1:10). Bar graphs represent the average slope ± SEM from the linear regression analyses described in Supplementary Fig. 5e. Data represent the mean ± SEM (n = 3 independent experiments). **p < 0.01 (−Clu vs. FLTau-YT:Clu 1:1 p = 0.0058); ***p < 0.001 (−Clu vs. FLTau-YT:Clu 1:10 p = 7.9 × 10⁻⁴) by one-way ANOVA with Bonferroni post hoc test. Lipofectamine was used as a transfection reagent.