FIGURE 3.

The majority of TDP-43 transfers occur from donor to acceptor cells (“anterograde”); however, there is a lesser degree of transfer from the acceptor to the donor cells (“retrograde”). A visualization of the Qdot-800 coculture model is provided in (A). Cells are differentiated in ECM gel with growth factors according to the scheme described. Prior to formation of the coculture, donor cells (shown here as GFP) were further labeled with Qdot-800 and selected by FACS to collect double-positive donor cells (+GFP and +Qdot-800) and plated directly onto fully differentiated acceptor cells. After 24 h of coculture, donor cells displaying retrograde transfer and acceptor cells displaying anterograde transfer were analyzed and separated using FACS and prepared for microscopy. Of the total transfer events, the majority of TDP-43 was transferred from donor to acceptor cells (anterograde; gray) (B,C, presented as proportions of detected transfer events). TDP-43 and TDP-43 truncated fragments also transferred from acceptor cells to donor cells (retrograde; black); however, retrograde transfer was comparatively minimal. Cell images corresponding to these data can be found in Supplementary Figure S4 (for data set B), and Supplementary Figure S5 (for data set C). Full ANOVA tables are provided in Supplementary Tables S5, S6. Data are presented as mean with significance determined by one-way ANOVA with Tukey post hoc test, n = 3–4 for each permutation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. The data represent the detected retrograde and anterograde transfer events as a proportion of all detected transfer events. BDNF, brain-derived neurotrophic factor; NRGβ1, neuregulin-β1; NGF, nerve growth factor; RA, retinoic acid; FACS, fluorescence-activated cell sorting.