Fig. 3.

Serum EVs from patients with SM are taken up by HSCs and promote their activation. EVs from 100 µL of individual serum samples in each group (HV, SM with tryptase <110 ng/mL, and SM with tryptase >110 ng/mL) were pooled together before addition to the cultured stellate cells. (A) Confocal images showing uptake of DiD-red fluorescently labeled EVs into HSCs. HSCs were incubated for 24 h with 100 µg DiD-labeled EVs (red) from the indicated groups. Nuclei from cells stained by DAPI are shown in blue and staining of α-SMA, a marker of HSC differentiation, in green. (Scale bar, 50 nm.) (B) Proliferation of HSCs in the presence of 10, 50, or 100 µg of EVs isolated from serum from HV or the indicated SM groups. EVs were added to cultured HSCs (in 500 µL of media) in 24-well plates for 24 h. Data represent mean ± SEM of three independent experiments. (C) Western blot analysis showing markers of HSC differentiation after 24-h incubation with 100 µg of the indicated EVs. The numbers underneath each band represent the average fold changes in fluorescence intensity normalized to actin and compared with the band intensity of untreated cells. The SD of fold changes for the three experiments was <10% of the mean. (D) Effect of SM-EVs on the release of the indicated cytokines by HSCs. HSCs were incubated with 100 µg of EVs of SM-EVs as above for 24 h. Cytokine levels released into the culture media were measured by ELISA, and the quantities released per well were then normalized by the total cellular protein content. *P < 0.05, **P < 0.01.