Fate and propagation of endogenously formed Tau aggregates in neuronal cells (2020)
Figure 2

Figure 2: Monitoring Tau seeding.

(A) Representative confocal images of DS1 cells (expressing soluble Tau RD-YFP), non-treated and treated with increasing concentrations of non-labelled K18 fibrils, fixed after two days and stained with WGA-Alexa 594 (red). The arrows point to examples of aggregate-containing cells, scale bars are 40 μm. On the right is the quantification of the mean percentage +SEM of cells where green aggregates were detected (the total number of cells analyzed over 3 independent experiments were 1188 for non-treated, 1552 for 0.3 μM, and 1506 for 1 μM), with statistical analysis by one-way ANOVA and Bonferroni post hoc test (****P = 6.20E-06 and 6.74E-05 for 0 to 0.3 and 0 to 1 comparisons respectively).

(B) Representative confocal images (40x) of RD-YFP SH cells, non-treated and treated with 1 μM K18-ATTO 594 fibrils, 48h after fibrils addition. Right insets are threefold enlargements of the boxed region of treated cells, merge and single channel pictures; white arrows point to partial colocalization. Scale bars are 10 μm, 2 μm in the insets. On the right is the quantification of the mean percentage (24%) +SD of cells where green aggregates were detected (2874 treated cells analyzed over 6 independent experiments), with statistical analysis by two-tailed unpaired t-test (****P = 3.75E-07).

(C) Relative proportion of DS9 cells expressing aggregates of Tau RD-YFP over a 50-hour period monitored by IncuCyte. DS9 cells were left untreated, the graph represents means of 3 independent experiments ±SD. See also Movie EV1.

(D) Monitoring the conversion of DS1 cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. Cells were plated, treated or not with K18-ATTO 594 fibrils at 1 μM (time 0) and placed into the IncuCyte incubator for six hours and next kept in culture for an additional 60 hours after medium change. The scatter plot represents means of 5 independent experiments (± SD for green and black curves). See also Movie EV2.

(E) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with K18 fibrils. Experiment and analysis were performed as in (D), the scatter plot shows means of 3 independent experiments ±SD for the green and black curves. See also Movie EV3.

(F) Monitoring the conversion of RD-YFP-expressing SH-SY5Y cells into inclusion-containing cells over a 70-hour period upon treatment with a cortex crude extracts from a AD-deceased patient. Experiment and analysis were performed as in (D), the scatter plot shows means of 2 independent experiments ±SD for the green curve. See also Movies EV4 and EV5. IncuCyte (20x objective) was set to acquire images every 30 minutes, phase-contrast and green channel (Excitation 440-480 nm, 400 ms) were acquired (9 images/well). Analysis was performed with IncuCyte software as in C-E to give the relative proportion of cells being converted to inclusion-containing cells (the end point of treated cells was arbitrarily set at 100%, black curve: non-treated cells, green curve: treated cells).

Data information: For (C-E), IncuCyte (20x objective) was set to acquire images every 30 minutes, phase-contrast, green channel (Excitation 440-480 nm, 400 ms) and red (excitation 565-605 nm, 800 ms) channels were acquired (4 images/well, 2 wells/condition). Analysis was performed with IncuCyte software to quantify: the number of red objects (K18-ATTO 594 fibrils, red curve: treated cells); the relative ratio between green objects and confluence, giving the relative proportion of cells being converted to inclusion-containing cells (the end point of treated cells was arbitrarily set at 100%, black curve: non-treated cells, green curve: treated cells); the relative ratio of green and red-colocalized objects over confluence (yellow curve: treated cells).