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Figure 1. Pore-forming protein βγ-CAT drives metabolite release from sEVs through channel formation
A: Colocalizations of βγ-CAT (green) and EV marker CD63 (red) in toad skin tissues were observed by laser confocal microscopy. Scale bar: 25 μm. B: Colocalizations of βγ-CAT (labeled with 5 nm colloid golden, black arrow) and EV marker CD63 (labeled with 10 nm colloid golden, red arrow) in toad skin were observed by TEM. Scale bar: 200 nm. C: Particle size distribution of sEVs isolated from BMDF (BMDF-sEVs) and L929 (L929-sEVs) cell culture medium by NTA. D: Morphological analysis of purified BMDF-sEVs and L929-sEVs by TEM. Arrows indicate EVs. Scale bar: 200 nm. E: Western blotting of purified BMDF-sEVs and L929-sEVs using EV-specific markers. F: Purified BMDF-sEVs (left) and L929-sEVs (right) were treated with βγ-CAT for 30 min, with appearance of βγ-CAT oligomers on treated EVs then determined by western blotting. G: Purified BMDF-sEVs (left) and L929-sEVs (right) were treated with PBS, LL-37, or βγ-CAT for 30 min, with ultra-morphology of EVs then observed by TEM. Scale bar: 200 nm. H: Heatmap from untargeted metabolomics of supernatants from βγ-CAT-treated and PBS-treated (control) BMDF-sEVs. Data are representative of five biological replicates. Data are related to Supplementary Table S1. I: KEGG pathway enrichment analysis of metabolites enriched in βγ-CAT-treated BMDF-sEV supernatants.
Supplementary Figure S1 Characterization of cultured B. maxima dermal fibroblast (BMDF) cell line
A: Diploid karyotype of cultured BMDF. B: Western blot detection of vimentin in cultured BMDF lysate.
Supplementary Figure S2 Strategy for isolation of BMDF and L929-derived EVs
Supplementary Figure S3 PCA score plot from untargeted metabolomics of supernatants from βγ-CAT-treated and PBS-treated (control) BMDF-EVs
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