Figure 7.

Tetracycline class antibiotics alter EV cargo via binding of the VPS4 protein of the ESCRT pathway. (a) U1 cells were pretreated with cART (45 µM) for 3 days followed by an additional dose of cART and tetracycline antibiotics (10 nM) for 5 days. Cells were lysed, and extracts were used for Western blot analysis for ESCRT proteins [ESCRT-I (TSG101),-II (EAP20, EAP45),-III (CHMP6), and exit (VPS4)] and Actin levels. (b) U1 cells were treated with a titration of (ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid) (EGTA) (0.1, 1, and 10 µM) for 5 days and whole cell extracts were analyzed by Western blot for ESCRT pathway proteins and Actin. (c) U1 whole cell extract was incubated with either biotin alone, or biotinylated Methacycline or Doxycycline. Resulting complexes were pulled down using streptavidin-sepharose beads. The samples were eluted with 40X excess free biotin, run on a 4–20% SDS/PAGE gel, and analyzed by Western blot for VPS4, VPS35/EAP45, and CHMP6. Densitometry counts as determined by ImageJ software show amounts of bound VPS4 relative to the U1 whole cell extract control (set to 100%).