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Fig. 3 | Cell Communication and Signaling

Fig. 3

From: A novel function of FAF1, which induces dopaminergic neuronal death through cell-to-cell transmission

Fig. 3

FAF1 positively controls exosome number. a SH-SY5Y cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium containing DMSO, GW4869 (10 μM), or monensin (10 μM), and the cells were cultured for 24 h. Left panel: CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Right panel: The graph shows the results of densitometric analysis of FAF1, Alix, and Hsc70 immunoblots in the CM shown in the left panel (n = 3). All lanes were loaded with the same amount of total protein. b Cells were transfected with siRNA against parkin or siRNA-resistant parkin construct. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. CL and concentrated CM were analyzed by western blotting with the indicated antibodies. Left panel: Representative western blots. Right panel: The graphs show the results of densitometric analysis of FAF1, Alix, and CD63 in CM, FAF1 and parkin in CL normalized to β-actin (n = 3). c Cells plated on 150 mm dishes were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with exosome-depleted medium, and the cells were cultured for 48 h. Then, exosomes were isolated from the CM with ExoQuick-TC. Left panel: Distribution profile of vesicles isolated by ExoQuick-TC. The blue line indicates exosomes from VC-transfected cells, and the red line indicates exosomes from 3xFlag-FAF1-transfected cells. Right panel: Exosome numbers were normalized to the final cell number (n = 3). The data are expressed as the mean ± S.D. of three independent experiments. Statistical comparisons were performed using ANOVA followed by Tukey’s HSD post hoc analysis (a and b) and Student’s t-test (c). *P < 0.05, **P < 0.01, ***P < 0.001, and n.s. = not significant

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