Fig. 4

Intracellular accumulation, escape to the cytosol and endogenous aggregation of various recombinant 2N4R Tau species. A The kinetics of intracellular accumulation of different Tau species, including large fibrils (L-fib), small fibrils (S-fib), oligomers (Oligo), fibrillization-derived monomers (F-mono), and recombinant monomers (Mono) within 48 h with 100 nM labeled Tau species. B Titration curve for intracellular accumulation of different Tau species after 20 h of incubation. Error bars represent SD; n = 3 per experimental condition. One-way ANOVA followed by a post-hoc test; ns: not significant, **p < 0.01, ***p < 0.001, ****p < 0.0001. C Schematic of the Tau entry assay. 0N4R P301S-Tau-HiBiT assemblies were added to cells expressing cytosolic LgBiT. Uptake of Tau assemblies may lead to cytosolic entry, resulting in Tau-HiBiT-mediated Nluc reconstitution by LgBiT binding. The addition of cell-permeable substrate results in the Nluc-mediated production of photons, which are readily quantifiable. Cytosolic entry is, therefore, proportional to the detected luminescent signal. D Percent of Tau-HiBiT that enters the cytosol of GPLN neurons following exposure to 2 ug/ml Tau-HiBiT monomers or Tau aggregated species (L-fib, S-fib, Oligo, Mono). Error bars denote SD. n = 3 per experimental condition. **p < 0.01; ***p < 0.001 by two-way ANOVA with Dunnett’s multiple comparisons. E Fluorescence microscopic images of HEK293-biosensor cells expressing P301S Tau-venus, either left untreated or treated with 200 nM unlabeled Tau small fibrils (S-fib). Arrows showing the inclusions of endogenous P301S Tau-venus. Scale bar: 61.7 nm. F Fluorescence analysis of cells treated with 200 nM Tau fractions and monomer using a fluorescence plate reader. Error bars represent SEM; n = 3 per experimental condition. One-way ANOVA followed by a post-hoc test; ns: not significant, **p < 0.01, ***p < 0.001 vs. Mono