Figure 3.

NPC1 is required for exosome cargo delivery. a, Cell surface expression of HAVCR1 in Huh7 parental (left panel, red dots) and NPC1 KO (right panel, green dots) cells stained with anti-HAVCR1 1D12 mAb and analyzed by flow cytometry. Data are representative of 3 independent experiments. b, Fluorescence of CMFDA-labeled Jurkat apoptotic cells bound to the cell surface of Huh7 NPC1 KO cells. Data are mean MFI ± sem, n=4 from 4 independent experiments. c, NPC1 is required for membrane fusion at endosomal compartments. Huh7 parental and NPC1 KO cells were incubated with R18-labeled liposomes for 0, 30, and 60 min at 37ºC and fusion (red) was observed by confocal microscopy. Cell nuclei counterstained with DAPI (blue). Data are representative of 3 independent experiments. Scale bar represents 20 μm. d,e, Huh7 parental and NPC1 KO cells treated for 24 h with exo-HAV or vpHAV produced in Huh7 cells infected with HAV.8Y-Bsd. d, Bsd-resistant CFU assay stained 10 days post-treatment with crystal violet (dark spots are Bsd-resistant colonies). Data are representative of 4 independent experiments. e, HAV RT-qPCR of total RNA extracted 48 h post-treatment. Data are mean ± sem, n=4 from 4 independent experiments. P values respect to parental exo-HAV were determined by one-way ANOVA test.