Fig. 5

SK1 was delivered by FMC-EV and required for the EV-induced activation of HSC. A Detection of SK1 expression in cell lysate and EV of FMC-1807 using Western blotting. B Detection of S1P in cell lysate but not in EV of FMC-1807 using Western blotting. C SK1 inhibitors abolished activation of LX-2 cell by FMC-EV. LX-2 cells were incubated with PBS or FMC-EV in the presence or absence of a SK1 inhibitor (CAY10621 or PF-543) for 24 h. The expression of α-SMA, which serves as an activation marker, in LX-2 cells was analyzed by Western blotting. The samples were analyzed in triplicate. Protein band intensity was quantified using ImageJ (lower panel). D Flow cytometric analysis of the phosphorylated STAT3 (p-STAT3) level in LX-2 cells after treatment with PBS (control), FMC-EV, or FMC-EV plus PF-543 (left panel). The mean fluorescence intensity of p-STAT3 is shown (right panel). E Flow cytometric analysis of the p-STAT3 level of LX-2 cells after treatment with PBS (control), FMC-EV, FMC-EV plus the endocytosis inhibitor (Chlorpromazine, 10 µg/ml)) or FMC-EV plus the dynamin inhibitor (Dynasore, 10 μM) for 24 h. F Flow cytometric analysis of the α-SMA level of LX-2 cells after treatment with PBS (control), FMC-EV, or FMC-EV plus a STAT3 inhibitor (Stattic) for 24 h (left panel). The mean fluorescence intensity of α-SMA is shown (right panel). n = 3 in each group. Quantitation data are presented as the mean ± SD. The Student’s t-test was used to determine the statistical significance