Figure 3.

CHMP6 knockdown accelerates the prion-like propagation of tau aggregation. FRET reporter cells or CRISPRi-HEK293T cells were transduced with individual sgRNAs targeting CHMP6 or VPS13D, or a nontargeting control sgRNA, and characterized for different phenotypes 5 days after transduction (A) Knockdown of CHMP6 and VPS13D does not impact steady-state levels of the tau-Clover2 construct in FRET reporter cells, as quantified by flow cytometry. B, individual gene knockdown does not impact uptake of tau fibrils. CRISPRi HEK293T cells were incubated with AF555-labeled tau fibrils for 1 h at 37 °C, stringently washed, and red fluorescence representing internalized tau fibrils was quantified by flow cytometry. The bar graph shows normalized fluorescence intensities and standard deviation of n = 3 technical replicates. C, CHMP6 knockdown accelerates prion-like propagation of tau aggregation. Representative fluorescence micrographs of the Tau.K18(LM)Clover2 reporter in cells 1 and 2 days after fibril addition are shown. Nuclei were counter-stained with Hoechst 33342. D, quantification of C. Tau aggregates were quantified by integrated density across the entire image and divided by total nuclei per image. Error bars represent mean ± S.D., where n = 3 images per condition (with at least 50 nuclei per image). *, p < 0.05; ***, p < 0.001 (two-tailed Student's t test for comparison to the values for nontargeting control sgRNA of the same day).