Fig. 5

Transferred FAF1 in adjacent cells maintains its death function. Donor cells were transfected with VC or 3xFlag-FAF1 plasmid. At 24 h after transfection, the culture medium was replaced with serum-free medium, and the cells were cultured for 24 h. a The CM was applied to recipient cells for 48 h. Heat-inactivated CM was boiled for 10 min. Cell death was determined by measuring PI uptake using a flow cytometer. b Rat donor primary neuronal cells were transduced with AAV1-hFAF1 viral vectors. At 3 days after transduction, the culture medium was replaced with serum-free neurobasal medium for 48 h. The CM was applied to recipient primary neuronal cells for 48 h. Cell death was determined by measuring PI uptake using a flow cytometer. c The CM from vector-transfected cells or FAF1-transfected cells containing vehicle (DMSO), z-VAD-fmk (100 μM), DPQ (30 μM), or Nec-1 (50 μM) for 48 h was applied to recipient cells. d After the CM was fractionated into the retained fraction (exosomal FAF1) and flow-through (vesicle-free FAF1), each CM fraction was applied to recipient cells for 48 h. e FAF1 was immunodepleted from CM with anti-FAF1 monoclonal antibody, and the CM was applied to recipient cells for 48 h. f After the recipient cells were pretreated with Dynasore (80 μM), heparin (200 μg/ml), or heparinase III (0.01 IU/ml) for 24 h, CM was applied to recipient cells for 48 h. Cell death was determined by measuring PI uptake using a flow cytometer. Data are expressed as the mean ± S.D. of three independent experiments (n = 3). Statistical comparisons were performed using ANOVA followed by Tukey’s HSD post hoc analysis. ^**P < 0.01, ^***P < 0.001, ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001