Figure 6: Inhibition of endo-lysosomal trafficking impairs FZD4 signalling. | Nature Communications

Figure 6: Inhibition of endo-lysosomal trafficking impairs FZD4 signalling.

From: Norrin-induced Frizzled4 endocytosis and endo-lysosomal trafficking control retinal angiogenesis and barrier function

Figure 6

(a) TOPFlash reporter assays in 293T cells transfected with the indicated constructs show that Dynasore inhibits Norrin/FZD4 signalling in a dose-dependent manner. (b) Co-expression of WT VPS4 has no effect on Norrin/FZD4 signalling, whereas dominant negative VPS4 EQ impairs Norrin/FZD4 signalling. Data are normalized to the first bar in each graph (n=3, mean+s.d. shown). Different signalling strength in a versus b is due to a shorter stimulation time in experiments where dynasore was added. (c–f) 3.5 h after induction of endocytosis, Norrin co-localized predominantly with LAMP1 in VPS4 expressing HeLa cells, and predominantly with EEA1 in VPS4 EQ expressing cells. In VPS4 EQ expressing cells, Norrin accumulation in early endosomes produced occasionally unusually large fluorescent puncta. (g,h) Quantification of co-localization (n=20, mean+s.d. shown, ***P<0.001). Scale bar, 10 μm.

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