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. 2022 May 6;39(5):110776. doi: 10.1016/j.celrep.2022.110776

Figure 3.

Figure 3

Defective endocytic machinery permits tau access to the cytosol in HEK-NGL cells

(A) Levels of entry of 50 nM tau-HiBiT assemblies applied to HEK-NGL cells after RAB5A or RAB7A depletion. Cells were treated with siRNA or non-targeting control (NTC) siRNA for 72 h before assaying tau entry; n = 3, N = 3 independent experiments.

(B) Western blot of cell lysates in (A), probing for RAB5, RAB7, and GAPDH.

(C–F) Entry of tau-HiBiT assemblies into HEK-NGL cells after depletion of VPS13D (C and D) or VPS35 (E and F) and confirmation via western blotting following the same experimental procedure described in (A) and (B).

(G) Fluorescence microscopy images of seeded tau-venus cells treated with NTC siRNA or VPS35-targeting siRNA for 72 h before addition of 250 nM exogenous tau assemblies in the absence of transfection reagents for another 72 h. Scale bars, 30 μm. White arrows indicate example fluorescent puncta.

(H) Quantification of seeded aggregation from the tau-venus seeding assays in (G); n = 6.

All error bars indicate mean ± SEM. ∗∗p < 0.01 by one-way ANOVA with Tukey's comparisons (C) or Kruskal-Wallis test with Dunn's comparisons (H). ∗∗∗∗p < 0.0001 by one-way ANOVA with Tukey's comparisons (A and E).