Figure 4
From: Rab35 and its effectors promote formation of tunneling nanotubes in neuronal cells
EHD1 is required for TNT formation: (a) Confocal images of cells transfected with GFP, GFP-EHD1. Arrows indicate TNTs. (b) Bar graphs representing the relative percentage of TNT-connected cells described in (a) (GFP = 100%, GFP-EHD1 = 150.5 ± 23%). (c) Bar graph showing the relative percentage of acceptor cells containing DiD-labelled vesicles from the co-cultures, where donor cells were transfected with GFP, GFP-EHD1 (GFP = 100%, GFP-EHD1 = 157.6 ± 5.5%). All graphs from three independent experiments and show mean ± SEM. (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001; by unpaired Student’s t-test); (d) Confocal images of cells transfected with shControl + GFP, shEHD1 + GFP, shEHD1 + GFP-EHD1. Arrows indicate TNTs. (e) Western blot of cells transfected with shRNA non-targeting (shCTL) or targeting EHD1 (shEHD1), showing the expression of EHD1 and α-tubulin as loading control. (f) Bar graphs representing the relative percentage of TNT-connected cells described in (a) (shControl + GFP = 100%, shEHD1 + GFP = 51.7 ± 12.7%, shEHD1 + GFP-EHD1 = 110.5 ± 8.2%). (g) Bar graph showing the relative percentage of acceptor cells containing DiD- labelled vesicles from the co-cultures, where donor cells were transfected with shControl + GFP, shEHD1 + GFP, shEHD1 + GFP-EHD1 (shControl + GFP = 100%, shEHD1 + GFP = 51.5 ± 9.0%, shEHD1 + GFP-EHD1 = 107.9 ± 5.6%). All graphs from at least three independent experiments and shows mean ± SEM. (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001; by one-way ANOVA with Tukey’s multiple comparison test). All bar graphs were analysed and represented using Graph Pad Prism version 7. Scale bars 10 µm.
