Fig. 4

Characterization of HLSC-derived EVs. a Representative Western blot analysis of ASS1 and CD63 expression in human liver stem cells (HLSCs) and the 10 K and 100 K fractions of HLSC-derived extracellular vesicles (EVs) obtained by differential ultracentrifugation. Tubulin was run on a parallel gel as a representative of loading (10 μg protein). Data represent one of two experiments with similar results. b Flow cytometric analysis of mesenchymal stem cell markers expressed by the 100 K fraction of HLSC-EVs. The 10 K fraction also expresses mesenchymal stem cell markers (data not shown). Data represent one of two experiments with similar results. c NanoSight quantification of HLSC-EVs obtained from the 100 K fraction before and after floating separation on iodixanol gradients (data represent a mean of three different experiments). Insert: representative electron microscopy of 100 K fraction of HLSC-EVs (scale bar = 100 nm). d NanoSight size distribution graph showing the quantity and size of HLSC-EVs obtained from the 15% iodixanol gradient. e Representative Western blot analysis of CD63 and ASS1 protein expression in HLSC-EVs isolated from different iodixanol fractions. Data represent one of two experiments with similar results. f Expression levels of ASS1 isofrom 1 (Iso1) and isoform 2 (Iso2) mRNA in 100 K HLSC-EVs. g Expression levels of ASS1 Iso1 mRNA in EVs isolated from different iodixanol fractions. h ASS1 mRNA Iso1 expression in EVs derived from ASS1 shRNA transfected HLSCs compared with EVs derived from HLSCs transfected with the scramble (SCR). Insert: representative Western blot analysis of ASS1 protein expression in EVs isolated from shRNA transfected HLSCs (EV shRNA-ASS1) and from EVs isolated from HLSCs transfected with the scramble shRNA (EV Ctrl-SCR). Data represent the mean of three independent experiments performed in triplicate. ^# p < 0.05, EV shRNA-ASS1 vs EV Ctrl-ASS1