Fig 1: Circulating liver derived transcripts predominantly exist outside extracellular vesicles (EVs) in the plasma of HCC patients.(A) EVs were isolated from the culture media of HepG2 and Huh7 liver cancer cells and characterized by western blotting. Equal amounts of protein samples were loaded and the enrichment of tetraspanin receptors CD9, CD63, CD81, Alix, and TSG101 was tested as markers of EVs. (B) EVs from either HCC, LC, or normal healthy control (NHC) plasma samples were extracted using ExoQuick kit and further purified using ultracentrifugation. In addition to EV markers Alix, CD63, and CD81, liver specific marker SERPINA1 was investigated. FAK was used as a loading control. (C) Characterization of EVs from various plasma samples was done by NTA NanoSight NS300 system and NTA software analysis was used to obtain the size distribution and quantitation of particles. (D) Plasma sample from an HCC patient (HCC101P) was fractionated into EV fraction and remaining EV-free fraction. RNAseq analysis was carried out on RNA from total plasma and its two fractions. Comparative expression profiles (TPM) of circulating transcripts within and outside EVs for more than 20 liver derived transcripts are shown. (E) Read piles of some of the liver specific transcripts shown above in 6D for whole patient plasma, EVs, and EV-free fraction highlight the differential expression. FTL and HBA1 show almost equivalent levels of transcripts within and outside EVs, but APOA2 and APOE preferentially exist outside EVs.