Fig. 4

NPC-derived exosomes enhance activation of YAP1 signaling in fibroblasts. A Exosomes released from HK1EBV cells contain FAPα protein. B Treatment of fibroblasts with HK1EBV cell-derived exosomes (10 μg/ml) increased levels of FAPα and active YAP1. Total protein lysates were harvested 1 and 3 hours post exosome treatment. GAPDH was used a protein loading control. C Immunostaining for active YAP1 (green) and FAPα (red) in fibroblasts treated with exosomes derived from HK1EBV cells. Cells were fixed 1 or 3 hours post exosome stimulation. Nuclei were stained with Hoechst 33342. Scale bar, 20 μm. D Quantification of active YAP1 expression in fibroblasts treated with exosomes compared with that in untreated cells. Cells were fixed 3 hours post exosome stimulation. Differential expression of active YAP1 was defined based on fluorescence intensities. Cell counts were determined using ImageJ software (***p < 0.001; chi-square test). E Assessment of mRNA expression of YAP1 downstream target genes. Total RNAs were extracted 3 or 6 hours after stimulating with HK1EBV-derived exoosmes. GAPDH was used as an internal control. Expression values were normalized to that of untreated fibroblasts. Values were expressed as mean and SD of two independent experiments. F Western blot analysis of YAP1 downstream proteins, CYR61, CTGF, and IGFBP3. Fibroblast protein lysates were harvested 1, 3, and 6 hours post exosome treatment. GAPDH was used as a loading control. G–I Immunofluorescence staining for YAP1 downstream molecules CYR61 (G), CTGF (H), and IGFBP3 (I) in fibroblasts treated with exosomes derived from HK1EBV cells. Cells were fixed 3 hours post exosome stimulation. Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Nuclei were stained with Hoechst 33342. Scale bars, 50 μm