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. 2020 Jun 19;12(6):665. doi: 10.3390/v12060665

Figure 4.

Figure 4

Figure 4

EV-associated c-Src activates HIV-1 in infected cells. (A) CEM EVs were isolated via ultracentrifugation (100K) and Western blotted for various src family kinases (lane 2). CEM WCE (lane 1) and actin were used as positive controls. (B) Log phase CEM cells were transfected with siRNA (100 nM) against c-Src for 72 h. EVs were isolated using ultracentrifugation (100K), measured, and then added to serum starved (1% FBS) U1 and ACH2 cells at a cell to EV ratio of 1:103, 1:104, or 1:105. Cells were collected 48 h later and Western blotted to assess the levels of newly synthesized p24. (C,D) ACH2 cells were transcriptional silence by serum starvation. This was followed by intracellular c-Src knockout in ACH2 cells by transfecting cells using siRNA against c-Src. CEM EVs (containing c-Src) were added to ACH2 cells at 0, 109, or 509 EVs (C) or 0 or 509 EVs (D) and allowed to incubate for 72 h at 37 °C. Samples were then analyzed by Western blot for HIV-1 Gag Pr55 (C), Gag p24 (D), and actin.