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. 2021 Sep 30;184(20):5089–5106.e21. doi: 10.1016/j.cell.2021.09.007

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© 2021 The Authors

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

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Figure S2 — Cell-to-cell contact favors fibrillar α-syn transfer between microglia and induces cytoskeletal changes, related to Figure 3

(A) Dose-response curve analysis for α-syn transfer capacities from donors to acceptors at increasing concentrations (0.1 – 1 μM). n = 4 with duplicate treatments for all conditions.

(B) Quantification of the ROS release of donor (right) and acceptor (left) cells with changing donor: acceptor ratios.

(C) Schematic drawing of staining strategy and quantification of the rate of acceptors engulfing dying donors (CellTracer labeled). The schematic was created using BioRender.com and Adobe Photoshop.

(D) Quantification of the transfer rate of fibrillar Amyloid-β between microglia. n = 3 with triplicate treatments for all conditions.

(E) Quantification of the transfer rate of fibrillar Tau between microglia. n = 3 with duplicate treatments for all conditions.

(F) Donors (CellTracer negative) and acceptors (CellTracer positive, blue) were co-cultured for 5 h and immunocytochemical analysis for Connexin 43 (Cx43) were performed.

(G) Donors and acceptors were co-cultured for the indicated time and the total length of the F-actin cytoskeleton of acceptors was measured.

(H–J) Quantification of the number of trunks (H), branches (I) and the mean trunk to branch end distance (J) of acceptor microglia over time. n = 4 per group. A total of at least 185-400 cells were analyzed.

(K) Donors and acceptors were co-cultured for the indicated time and the total length of the F-actin cytoskeleton of donors was measured.

(L–N) Quantification of the number of trunks (L), branches (M) and the mean trunk to branch end distance (N) of donor microglia over time. n = 4 per group. A total of at least 185-400 cells were analyzed.

Graphs in A-E are presented as mean ± SEM and were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post hoc test. Graphs in G-N are presented as violin plots and were analyzed by one-way ANOVA followed by Tukey’s multiple comparison post hoc test. ^∗∗∗∗p < 0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01 compared to 0 h.

Scale bars: 5 μm.