Figure 5.

PHEV activates autophagosome formation but prevents its fusion with lysosomes. (A) Neuro-2a cells were transfected with mRFP-GFP-LC3. As the positive control for induction of autophagy, Neuro-2a cells were transfected with mRFP-GFP-LC3 and then treated with complete medium supplemented with 200 μM rapamycin for 3 h. At 0, 12, and 24 h post-transfection, the cells were fixed and assessed with GFP and mRFP fluorescence. Scale bars: 50 μm. (B) Neuro-2a cells were used to analyze the colocalization of LysoTracker-stained acidified vesicles and GFP-LC3-positive autophagosomes in the mock-infected and PHEV-infected cells at 12, 24, 36, and 48 h. Representative images are shown in Figure 5B. (C) Furthermore, the fusion of autophagosomes with lysosomes was analyzed as colocalization of the autophagosome marker GFP-LC3 with the lysosome marker LAMP1. Nuclear DNA was stained with DAPI. One of the three experiments conducted is shown. (D) Western blotting analysis of LAMP1 changes in PHEV-infected cells for 12, 24, 36, and 48 h were shown in Figure 5D, compared with the relevant times in mock cells. Scale bars: 10 μm.