Fig. 1. Lipotoxic hepatocyte-derived EVs are enriched with active ITGβ₁.

(A) Top ranked canonical pathways identified by IPA of proteomic data on EVs derived from vehicle or LPC-treated PMH. Immunoblot analysis showing protein levels of integrin family members and Talin-1 on EVs and whole cell lysate (WCL) from (B) PMH or (C) Huh7 cells treated with vehicle or 20 μM LPC for 4 hours. Beta-actin, and the EV markers TSG101, CD63 and CD81 were used as loading controls for WCL and EVs, respectively. (D) Immunogold electron microscopy images showing immunoreactivity for ITGβ₁ in an active conformation on EVs derived from PMH treated with vehicle (Veh-EV) or LPC (LPC-EV). Nanoscale flow cytometry showing expression levels of active ITGβ₁ on EVs, (E) various sizes silica nanoparticles used as calibration beads to define EVs based on the particle size (top panel). ITGβ₁⁺ EVs from PMH treated with Veh or LPC (bottom panel), quantification of ITGβ₁-positive EVs from (F) PMH and (G) Huh7. Bar columns represent mean ± standard error of the mean (SEM); n=3–5. (H) Quantification of ITGβ₁⁺ EVs in the serum of patients with simple steatosis (n=8), and NASH with stage 1–2 fibrosis (n=17). Graphs represent mean ± SEM; *p<0.05, **p<0.01 (Unpaired t test).