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. 2017 Oct 9;8(59):99841–99860. doi: 10.18632/oncotarget.21668

Figure 6. EVs-mediated complement activation is associated with the alteration of the endogenous C3 and properdin.

Figure 6

(A) EVs from KSHV-infected HUVECs are not a direct activator for the complement system. HUVECs treated with EVs from KSHV-infected HUVECs at 24 hpi for different lengths of time were examined for C5b-9 deposition. After exposure to the EVs for the indicated durations, the cells were treated with NHS for another 30 min, and analyzed for C5b-9 depositions by IFA. Scale bar: 50 μm. (B) Endogenous C3b and properdin were detected on the HUVECs treated with EVs from KSHV-infected cells. Naïve HUVECs were treated with EVs from mock- or KSHV-infected HUVECs for 24 h, followed by analyzing for C3b and properdin on the cell surface by IFA. Scale bar: 10 μm. (C) Western blotting for C3b and properdin. Naïve HUVECs were treated with EVs from mock- or KSHV-infected HUVECs, and cell lysates were prepared from these cells with or without treatment with NHS for 30 min. β-actin was used as a loading control. Mock: cell lysate of HUVECs treated with EVs from mock-infected cells, KSHV: cell lysate of HUVECs treated with EVs from KSHV-infected cells. (D) Cell ELISA for the depositions of C5b-9 with single complement factor-depleted human serum. Naïve HUVECs were treated with EVs from mock- (Mock Evs) or KSHV-infected cells (KSHV EVs) followed by exposure for 30 min to human serum with depletion of the indicated individual complement factor with and without the addition of the respective purified complement protein. Then C5b-9 depositions were quantified by cell ELISA. Results are shown as mean ± SD, N = 5, **p < 0.01.